-
[
International C. elegans Meeting,
1993]
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[
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.
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[
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).
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[
International C. elegans Meeting,
1995]
We are using free-living nematodes to identify genes likely to be responsible for ivermectin resistance in parasitic nematodes. Previous studies revealed i) that low-level (5-15X) resistance in C. elegans is very common, conferred by recessive mutations in >30 genes, ii) that dominant resistance is less frequent, we find 6,000 recessives for each dominant mutation and iii) that high level resistance (>100X) is very rare, requiring recessive mutations in more than one gene. We observed a similar pattern of resistance in other free-living nematodes. Peter Hunt and Warwick Grant discovered that most low level resistance mutations also confer defects in dye-filling of the amphid neurons (Johnson, et al. WBG 13(3), 70). Many ivermectin resistance mutations are now known to be alleles of che, osm and dyf genes that control the dye-filling phenotype (Starich et al (1995) Genetics 139:171). Complementary results with other species show: i) that resistance in species that dye-fill is accompanied by dye-filling defects and iii) that species that do not dye-fill are "naturally" resistant to ivermectin. We are currently studying two new forms of resistance: 1) Dyf(+) low level resistance -- some mutations are unusual alleles of known genes -- and 2) mid-level (20-40X) resistance - - some mutations are in genes that contribute to high level resistance, other mutations are in new genes. Finally, we have been measuring the ivermectin resistance allele frequency in wild nematode populations. In one Dfy(+) diecious nematode species (not yet identified) we isolated 4 resistant strains (all Dyf) from the inbred progeny of 93 fertilized females -- apparent allele frequency in the wild: ~0.01 (4/93x4).
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[
International C. elegans Meeting,
1993]
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[
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.
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[
International C. elegans Meeting,
1997]
Mutations which also confer a Dyf phenotype are a major cause of resistance to ivermectin and related macrocyclic lactone anti-nematode drugs, the Avr, for avermectins resistance, phenotype. Many of the same mutations also confer a DafD phenotype that can, under certain circumstances, be suppressed by mutations in
unc-104 and
unc-116 which affect neurotransmitter release (Vowels & Thomas, WBG12(2):85). We have further examined the interactions between mutations with DyfAvr phenotypes with
unc-104,
unc-116, and also with
snt-1, another locus that affects neurotransmission. We have observed allele specific suppression of the Avr phenotype in some double mutants, uncoupling the Dyf and Avr phenotypes. We have also identified and characterised rare nonDyf Avr alleles of some genes that commonly produce DyfAvr mutations. These results suggest that resistance to macrocyclic lactones is not due simply to a failure of drug entry via the amphids. Instead they imply that resistance may require a signal from the amphid neurones. The signal might, for example, be part of a sensory pathway that links amphid neurones with the pharynx. Blockage of pharyngeal pumping is correlated with drug action and the pharyngeal motorneurone M3 is one well characterised site at which ivermectin acts (Avery & Dent, WBG 13(4):72). Screens for suppressors of the Avr phenotype are expected to uncover additional components of this putative sensory transduction pathway. Preliminary results from such screens have already yielded new mutants. Most have no visible phenotype. A model of the action of ivermectin and related macrocyclic lactones incorporating these results will be presented.
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[
Proc Natl Acad Sci U S A,
1999]
Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene
sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene
hop-1 and to examine possible genetic interactions between
hop-1 and
sel-12, we used a reverse genetic strategy to isolate deletion alleles of both loci. Animals bearing both
hop-1 and
sel-12 deletions displayed new phenotypes not observed in animals bearing either single deletion. These new phenotypes-germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects-resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of
hop-1 and
sel-12 single and double mutant animals suggest that
sel-12 provides more presenilin function than does
hop-1.
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[
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