-
[
International C. elegans Meeting,
1993]
-
[
Midwest Worm Meeting,
1998]
Direct PCR screening of chemically mutagenized nematode populations has rapidly become a method of choice for obtaining deletion mutations in a targeted gene. NemaPharm has utilized high-throughput techniques based on liquid cultures and microtiter plate arrays to obtain over 70 deletion mutations to date. Following mutagenesis treatment of P0 nematodes, each well of a 96-well microtiter plate is seeded with ~20 F1 worms, which are grown in liquid cultures to produce about 2000 F2 larvae. One-third of the worms from each well are removed to produce a matching genomic DNA array as well as a plate DNA pool of ~4,000 mutagenized genomes, and the residual worm plates are frozen. In the PCR screen, we design nested primer pairs ~3 kb apart to preferentially target proximal exon-rich regions encoding critical domains. The first round of PCR is performed on plate pools in 96-well PCR format; each 96-pool screen thus samples about 400,000 genomes. Amplicons smaller than the wild-type amplicon represent candidate deletions. Candidate pools are re-sampled in quadruplicate to eliminate false positives, which in our experience constitute about three-fourths of the first-round candidate deletions. The plate DNA array is then screened to identify a specific library address. Before thawing the well address, we map the deletion by restriction enzymes and use this information to design three new PCR primers, A and B which flank the deletion (and are used to sequence the exact breakpoints) as well as a third primer C from within the deletion. We perform the sib selection by cloning worms recovered from the thawed well into microtiter plate cultures. Following growth in liquid media, an aliquot is removed for 96-well PCR using primers A and B in a single round of PCR to identify clones bearing the deletion. Positive lines are then transferred to agar plates, and multiplex single worm PCR using primers A,B, and C is then used to distinguish homozygous from heterozygous animals in subsequent generations. We have identified deletions from worm populations mutagenized with EMS, ENU, diepoxyoctane and UV/trimethylpsoralen. Among our first 72 deletion mutations, the average deletion size was approximately 1300bp +/- 500bp SD, and somewhat smaller for EMS (1093 +/- 340bp) compared to the other three mutagens. The average number of viable worms recovered from the frozen wells was 319 +/- 249 (range 16-1000). A small number of thawed library wells failed to yield the expected deletion mutant, and the failure rate was >50% when <150 viable animals were recovered. Currently we recover deletions in about 2/3 of targeted loci from a cumulative library of 1 million genomes, a figure which may underestimate the number of potential detectable deletions since further screening is not pursued after identifying a deletion. In any case, the frequency of deletions obtained from each of the 4 chemical treatments far exceeds the predicted spontaneous rate of deletions of this size range.
-
[
West Coast Worm Meeting,
1998]
Direct PCR screening of chemically mutagenized nematode populations has rapidly become the method of choice for obtaining deletion mutations in a targeted gene. NemaPharm has used high-throughput techniques based on liquid cultures and microtiter plate arrays to obtain over 70 deletion mutations to date. Following mutagenesis treatment of P0 nematodes, each well of a 96-well microtiter plate is seeded with ~20 F1 worms, which are grown in liquid cultures to produce about 2000 F2 larvae. From each well, equal numbers of worms are used to produce a matching genomic DNA array, a plate DNA pool of ~4,000 mutagenized genomes, and a frozen stock. For the PCR screen, we design nested primer pairs 2.8-3.2 kb apart and preferentially target proximal exon-rich regions encoding critical domains (if known). The first round of PCR is performed on 96 plate pools in a microtiter-plate format. Amplicons smaller than the wild-type amplicon represent candidate deletions. Candidate pools are re-sampled in quadruplicate to eliminate false positives, which in our experience constitute about three-fourths of the first-round candidate deletions. The plate DNA array is then screened to identify a specific library address. Before thawing the well address, we map the deletion by restriction enzymes and use this information to design three new PCR primers, two primers which flank the deletion (and are used to sequence the exact breakpoints) and a third primer from within the deletion. We perform the sib selection by cloning worms recovered from the thawed well into microtiter plate cultures. Following growth in liquid media, an aliquot is removed for 96-well PCR using flanking primers in a single round of PCR to identify clones bearing the deletion. Positive lines are then transferred to agar plates, and multiplex single worm PCR using both the flanking and internal primers is then used to distinguish homozygous from heterozygous animals in subsequent generations. We have identified deletions from worm populations mutagenized with EMS, ENU, diepoxyoctane and UV/trimethylpsoralen. Among our first 72 deletion mutations, the average deletion size was approximately 1300 500 bp; EMS may generate slightly smaller deletions with a tighter size distribution compared to the other three mutagens (1093 340 bp vs. 1359 562 bp). The average number of viable worms recovered from the frozen wells was 319 249 (range 16-1000). A small number of thawed library wells failed to yield the expected deletion mutant, and the failure rate was >50% when <150 viable animals were recovered. The frequency of deletions obtained from each of the four mutagens far exceeds the predicted spontaneous rate of deletions of this size range (estimated by Phil Anderson to be approximately 1 in 100-200 million genomes). Currently we routinely screen libraries representing one million genomes, and are successful in isolating deletion mutants for about two of every three targeted loci; this rate of success may underestimate the number of potentially detectable deletions since further screening is not pursued after identifying a deletion. If we fail to identify a deletion in the initial screen, we screen additional libraries representing typically another 600,000 genomes; from the combined screen of 1.6 million genomes, we have been able to identify deletion mutants for almost all loci we have targeted.
-
[
East Coast Worm Meeting,
1998]
Direct PCR screening of chemically mutagenized nematode populations has rapidly become a method of choice for obtaining deletion mutations in a targeted gene. NemaPharm has utilized high-throughput techniques based on liquid cultures and microtiter plate arrays to obtain over 70 deletion mutations to date. Following mutagenesis treatment of P0 nematodes, each well of a 96-well microtiter plate is seeded with ~20 F1 worms, which are grown in liquid cultures to produce about 2000 F2 larvae. One-third of the worms from each well are removed to produce a matching genomic DNA array as well as a plate DNA pool of ~4,000 mutagenized genomes, and the residual worm plates are frozen. In the PCR screen, we design nested primer pairs 2.8-3.2 kb apart to preferentially target proximal exon-rich regions encoding critical domains (if known). The first round of PCR is performed on plate pools in 96-well PCR format; each 96-pool screen thus samples about 400,000 genomes. Amplicons smaller than the wild-type amplicon represent candidate deletions. Candidate pools are re-sampled in quadruplicate to eliminate false positives, which in our experience constitute about three-fourths of the first-round candidate deletions. The plate DNA array is then screened to identify a specific library address. Before thawing the well address, we map the deletion by restriction enzymes and use this information to design three new PCR primers, A and B which flank the deletion (and are used to sequence the exact breakpoints) as well as a third primer C from within the deletion. We perform the sib selection by cloning worms recovered from the thawed well into microtiter plate cultures. Following growth in liquid media, an aliquot is removed for 96-well PCR using primers A and B in a single round of PCR to identify clones bearing the deletion. Positive lines are then transferred to agar plates, and multiplex single worm PCR using primers A,B, and C is then used to distinguish homozygous from heterozygous animals in subsequent generations. We have identified deletions from worm populations mutagenized with EMS, ENU, diepoxyoctane and UV/trimethylpsoralen. Among our first 72 deletion mutations, the average deletion size was approximately 1300bp +- 500bp SD, and somewhat smaller for EMS (1093 +- 340bp) compared to the other three mutagens. The average number of viable worms recovered from the frozen wells was 319 +- 249 (range 16-1000). A small number of thawed library wells failed to yield the expected deletion mutant, and the failure rate was >50% when <150 viable animals were recovered. Currently we recover deletions in about 2/3 of targeted loci from a cumulative library of 1 million genomes, a figure which may underestimate the number of potential detectable deletions since further screening is not pursued after identifying a deletion. In any case, the frequency of deletions obtained from each of the 4 chemical treatments far exceeds the predicted spontaneous rate of deletions of this size range (estimated by Phil Anderson to be approximately 1 in 100-200 million genomes).
-
[
Zootaxa,
2022]
Rhagovelia medinae sp. nov., of the hambletoni group (angustipes complex), and R. utria sp. nov., of the hirtipes group (robusta complex), are described, illustrated, and compared with similar congeners. Based on the examination of type specimens, six new synonymies are proposed: R. elegans Uhler, 1894 = R. pediformis Padilla-Gil, 2010, syn. nov.; R. cauca Polhemus, 1997 = R. azulita Padilla-Gil, 2009, syn. nov., R. huila Padilla-Gil, 2009, syn. nov., R. oporapa Padilla-Gil, 2009, syn. nov, R. quilichaensis Padilla-Gil, 2011, syn. nov.; and R. gaigei, Drake Hussey, 1947 = R. victoria Padilla-Gil, 2012 syn. nov. The first record from Colombia is presented for R. trailii (White, 1879), and the distributions of the following species are extended in the country: R. cali Polhemus, 1997, R. castanea Gould, 1931, R. cauca Polhemus, 1997, R. gaigei Drake Hussey, 1957, R. elegans Uhler, 1894, R. femoralis Champion, 1898, R. malkini Polhemus, 1997, R. perija Polhemus, 1997, R. sinuata Gould, 1931, R. venezuelana Polhemus, 1997, R. williamsi Gould, 1931, and R. zeteki Drake, 1953.
-
[
Journal of Microscopy,
1973]
Nematodes and mildew-infected barley leaves when examined in the scanning electron microscope after critical point drying (CPD) from sulphur dioxide (critical temperature 157.7C) showed no obvious physical damage, but the specimens had a surface deposit which was probably heat damaged natural waxes. The nematode Caenorhabditis elegans and clover roots (Trifolium subterraneum) showed no physical or heat damage after CPD from monochlorodifluoromethane (Freon 22, critical temperature 96C). The hyphae and conidia of unfixed mildew on barley were damaged after CPD from Freon 22, probably due to the Freon extracting lipids from the cell walls. Freon 22 was preferred for most specimens as it is cheap, easy to get
-
[
International C. elegans Meeting,
1999]
For generating a large number of separate populations of worms, liquid cultures in the wells of microtiter plates use space more efficiently than do standard cultures on agar plates. At NemaPharm, we routinely grow worms in microtiter plates for a wide variety of procedures including both forward and reverse genetics experiments as well as for high-throughput chemical screening. To automate distribution of worms into microtiter plate wells, we have constructed a machine that efficiently sorts and dispenses live nematodes. The mechanism used for measuring nematodes is based, in principle, on the nematode counting machine built by Lou Byerly, Randy Cassada and Dick Russell in the early 1970's (1). Nematodes are suspended in liquid and passed though a narrow nozzle into the center of a rapidly flowing sheath current. The resultant shear forces straighten the animals and orient them parallel to the direction of flow. The straightened animals are then passed at high speed (~1 m/sec) through an orthogonal sheet of laser light where, in the current model, an in-line detector measures the attenuation of the laser light and an attached computer calculates the length of the nematode based upon time-of-flight through the beam. Below the detector, the worm-containing stream is deflected to waste by a air jet controlled by the computer that shuts off momentarily to deliver a selected animal to a well (distribution volume is 0.25 - 1 microl per worm). The current sorter/dispenser is capable of distributing 10 worms to the wells of 96-well plates in 2 minutes with a precision of +/-0.7 worms. Distribution to 384 and 1536 well plates is also feasible with the current model. Machines with more sophisticated detecting and measuring capabilities are planned for the future. (1) Byerly, et. al. (1975) Rev Sci. Instrum., 46 :517-522
-
[
J Biol Chem,
1990]
The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by noncovalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pI values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (approximately 6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5'-flanking region has no TATA box and contains preferred and minor transcription start sites.
-
[
Worm Breeder's Gazette,
1994]
R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with
bc1-2, the human homolog to
ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and
bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar
lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with
ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.
-
[
Nat Commun,
2021]
R-bodies are long, extendable protein polymers formed in the cytoplasm of some bacteria; they are best known for their role in killing of paramecia by bacterial endosymbionts. Pseudomonas aeruginosa PA14, an opportunistic pathogen of diverse hosts, contains genes (referred to as the reb cluster) with potential to confer production of R-bodies and that have been implicated in virulence. Here, we show that products of the PA14 reb cluster associate with R-bodies and control stochastic expression of R-body structural genes.PA14 expresses reb genes during colonization of plant and nematode hosts, and R-body production is required for full virulence in nematodes. Analyses of nematode ribosome content and immune response indicate that P. aeruginosa R-bodies act via a mechanism involving ribosome cleavage and translational inhibition. Our observations provide insight into the biology of R-body production and its consequences during P. aeruginosa infection.