Clover R et al. (1993) International C. elegans Meeting "Tools for identifying homologues of C. elegans genes."

Clover R, Johnson CD

[International C. elegans Meeting, 1993]

Clover R et al. (1998) Midwest Worm Meeting "High-Throughput Isolation of Deletion Mutants of C. elegans"

Chen JS, Liu LX, Clover R, Spoerke JM, Mulligan EL, Armstrong BL, Hardiman G

[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.

Clover R et al. (1998) East Coast Worm Meeting "High-Throughput Isolation of Deletion Mutants of C. elegans"

Chen JS, Hardiman G, Clover R, Mulligan EL, Liu LX, Armstrong BL, Spoerke JM

[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).

Clover R et al. (1998) West Coast Worm Meeting "High-Throughput Isolation of Deletion Mutants of C. elegans"

Clover R, Chen JS, Basson M, Liu LX, Spoerke JM, Armstrong BL, Hardiman G, Mulligan EL

[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.

CD Johnson et al. (1999) International C. elegans Meeting "Automated sorting and dispensing of C. elegans to wells of microtiter plates"

CD Johnson, R Clover, B Reardon, AA Ferrante, WP Hansen, PB Krauledat

[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

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Duke A, Duerr JS, McManus JR, Rand JB, Moulder GL, Kohn RE, Barstead RJ

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, H H Rees, R A Clegg, M J Fisher, M Tabish

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zelmanovich, V., Livshits, L., Zuckerman, B., Smith, Y., Gross, E., Abergel, R., Romero, L., Abergel, Z.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Rubin CS, Angelo RG

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.

Hicks, Tara et al. (2021) International Worm Meeting "R-loop-induced irreparable DNA damage in C. elegans meiosis"

Smolikove, Sarit, Williams, Cameron, Koury, Emily, Hicks, Tara

[International Worm Meeting, 2021]

Most DNA-RNA hybrids are formed naturally during transcription and are composed of a nascent RNA strand hybridized to DNA as part of R-loops. The accumulation of these structures in S-phase can result in replication-transcription conflict, an outcome which can lead to the formation of double strand breaks (DSBs). While R-loops' role in mitotically dividing cells has been characterized, there are only a handful of studies describing the effect of R-loops in meiosis and these studies present a complex picture of the outcome of R-loop formation on germ cells. Here we show that DSBs formed by R-loops trigger an altered cellular response to DNA damage. RNase H is an enzyme responsible for degradation of the RNA strand in DNA-RNA hybrids and plays an essential role in preventing this outcome and its deleterious consequences. Using null mutants for the two Caenorhabditis elegans genes encoding for RNase H1 and RNase H2 (hereby rnh mutants), our studies explore the effects of replication stress-induced DNA-RNA hybrid accumulation on meiosis. As expected, rnh mutants exhibit an increase in R-loop formation. Consequently, an elevation of DSBs in germline nuclei is evidenced by the accumulation of RAD-51 foci. Despite no repair mechanism abrogation, rnh mutants fail to repair all DSBs generated, leading to a fragmentation of chromosomes in diakinesis oocytes. By combining our double mutant with a spo-11 null mutation, we show that although replicative defects are the main contributor to the phenotype, R-loops formed in meiosis are likely contributors as well. We present evidence that while rnh mutants accumulate DNA-RNA hybrids and subsequent DSBs may signal a degree of checkpoint activation in mitosis, some damaged nuclei prevail past the checkpoint, enter into meiosis, and remain unrepaired throughout. Moreover, we find no evidence of an increase in apoptosis, which indicates that DNA damage generated by R-loops remain undetected by an apoptotic checkpoint. This data altogether points to DSBs initiated by R-loops representing an irreparable type of DNA damage that evades cellular machineries designed for damage recognition.