9 Papers found (0.009 seconds)

Praitis, Vida et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Characterization of a gene important for C. elegans gastrulation"

Liszewski, Walter, Mushi, Juliet, Miller, Michael, Schacht, Angela, Imlay, Leah, Praitis, Vida, Yohannes, Lensa, Feddersen, Charlotte, Sullivan-Wilson, Alexander, Kniss, Sarah, Ha, Dae Gon, Chakravorty, Adityarup, Yilma, Zelealem, Tyson, Stock

[

C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,

2010]

In C. elegans , gastrulation begins with ingression of E blastomeres at the 26-cell stage and ends with the establishment of a mature vermiform embryo 6 hours later (Sulston et al 1983). While significant research has been done on the initiation of gastrulation, ingression of cells in mid-gastrulation is not as well understood. What is clear is these later cells, derived from very distinct lineages, must acquire specific fates and positional information to ingress properly (Nance, et al 2005; Rohrschneider & Nance 2009). Using a genetic approach, my research group has been characterizing a gene important during mid-gastr ulation. We have identified ru5 , a conditional embryonic lethal mutation that causes terminal phenotypes that include body and head enclosure failure, a detached pharynx, and other body morphogenesis defects. Temperature shift experiments indicate that the gene acts during gastrulation. The ru5 mutation does not cause major changes in cell fate or position, as expression of six tissue-specific markers is normal. We do observe changes in the expression of vab-7/even-skipped homeodomain protein. The defects in vab-7 expression caused by ru5 appear to be distinct from those caused by tbx-8/9 mutants (Pocock, 2004; Andachi, 2004) indicating that the ru5 allele helps to define a second regulatory pathway for vab-7 . We have mapped the ru5 locus to ~19 on L.G.I and are continuing experiments to identify the altered gene. We have also begun a genetic suppressor screen to identify interacting genes, using the mos1 transposon as a mutagen (Bessereau, 2001). We wi ll report progress in these areas at the meeting.

James L Lissemore et al. (2003) International Worm Meeting "Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene."

Laura L Lackner, George D Fedoriw, James L Lissemore

[

International Worm Meeting,

2003]

PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise in a molecular biology lab course during the Spring 2003 semester and their results will be presented. We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains used in this exercise.

James L Lissemore et al. (2001) International C. elegans Meeting "Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene"

James L Lissemore, George D Fedoriw, Laura L Lackner

[

International C. elegans Meeting,

2001]

PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise in a molecular biology lab course during the Spring 2001 semester and their results will be presented. We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains and primers used in this exercise.

Johnson, Cheryl et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Maternal-effect epigenetic germ-line silencing of fem-1"

Johnson, Cheryl, Spence, Andrew

[

C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,

2010]

Several mechanisms maintain the integrity of the C. elegans germ line by restricting the expression of inappropriate sequences. We describe a previously uncharacterized form of silencing that can be prevented by maternally inherited transcripts. We discovered this phenomenon while studying fem-1 , a gene that is required for all aspects of male development. When females carrying any of three deficiency alleles, fem-1(Df) , are crossed to wild-type males, their heterozygous progeny often exhibit germ-line feminization (the Fog phenotype). This feminization is not observed in the cross-progeny of animals carrying any other fem-1 alleles, including the null allele e2268 . Injecting fem-1 RNA, even if mutated to eliminate coding potential, into the syncytial germ line of fem-1(Df) females can rescue the Fog phenotype of their offspring, further suggesting that there is a maternal requirement of fem-1 RNA for spermatogenesis in the zygote. Genetic and molecular evidence demonstrate that the germ-line feminization is caused by a reduction in zygotic germ-line fem-1 activity and is meiotically heritable. We propose that fem-1 is heritably silenced in the germ line in the absence of maternal RNA, but provision of maternal fem-1 transcripts can promote activity of the gene, effectively licensing its expression. An RNAi screen for modifiers of this maternal-effect regulation implicated small RNA pathways in both the silencing and licensing components of this phenomenon. Perhaps fem-1 is targeted for silencing by certain RNAs, and the role of licensing may be to oppose or inactivate them with maternal fem-1 RNA. We are investigating whether specific sequences in or near fem-1 are needed for its susceptibility to silencing. Alternatively, this form of silencing may act in the germ line more generally wi thout distinguishing between loci based on specific characteristics in their sequences.

JL Lissemore et al. (1999) International C. elegans Meeting "Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene"

JL Lissemore, LL Lackner, GD Fedoriw

[

International C. elegans Meeting,

1999]

PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise during the Spring 1999 semester. While there was variation in the yield of the products, especially with primer pair B, all five groups detected the presence of deletions of the expected size in the expected strains. We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains and primers used in this exercise. LLL and GDF made equivalent contributions to this work.

Shiraishi, Hirohisa et al. (2013) International Worm Meeting "Genetic requirements of the pentose phosphate pathway for the intestinal granule formation in C. elegans."

Shiraishi, Hirohisa, Asanuma, Yumi, Tanabe, Mari, Oikawa, Takato, Aoyama, Reiko, Nishikori, Kenji, Ohashi-Kobayashi, Ayako, Ishibashi, Maya, Tanji, Takahiro

[

International Worm Meeting,

2013]

We have focused on intestinal granular organelles observed from late larval to early adult stage in C. elegans since a major subset of them dynamically emerge or evanesce in an age- and nutrient-dependent manner (Nishikori K., et al., C. elegans Topic Meeting 2012, Madison, WI). Such organelles are non-acidic granules to which HAF-4 and HAF-9, ABC transporters homologous to a mammalian lysosomal peptide transporter ABCB9, co-localize. The deletion mutants for haf-4 and haf-9 exhibited the loss of the HAF-4/HAF-9-positive granules and some physiological defects such as slow growth and reduced brood size, suggesting that the granules play important roles in the storage and supply of some nutritional components during reproductive period. To uncover the physiological roles and the regulatory mechanisms of the intestinal granules, we have performed comprehensive RNAi screening using C. elegans RNAi Feeding Library (Open Biosystems). To achieve the quick and reliable evaluation of RNAi effects, we adopted a high-powered digital microscope for observation. This system magnifies live worms on the rearing agar plate up to 5,000 times, which facilitates screening for the presence of intestinal granules. So far several tens of candidates with reduced granular number have been identified among approximately 2,000 genes tested. In this meeting, we report tkt-1, an orthologous gene for mammalian transketolase (TKT), as one candidate. TKT acts in the pentose phosphate pathway (PPP), which contributes to fatty acid synthesis and oxidative stress prevention via NADPH production as an alternative to glycolysis pathway. We found that RNAi of some PPP-related genes and the glutathione-disulfide reductase gene results in the reduction of the HAF-4::GFP-positive granules and the DHS-3::GFP-positive lipid droplets, but not of the GLO-1::GFP-positive lysosome-related organelles. The NADPH-producing oxidative pathway of PPP might be necessary for the normal formation of specific subsets of the intestinal granules in C. elegans.

Anupom, T. et al. (2017) International Worm Meeting "A compact and modular lifespan machine for aging studies in Caenorhabditis elegans."

Szewczyk, N., Anupom, T., Gupta, S., Driscoll, M., Rahman, M., Vanapalli, S., Blawzdziewicz, J.

[

International Worm Meeting,

2017]

The lifespan of an organism is the ultimate indicator of the molecular mechanisms active during aging of an organism. Short lifespan, well-established genetics, and conserved signaling pathways make the nematode Caenorhabditis elegans an attractive model for aging research. Large-scale automated screens for longevity genes in the nematode C. elegans often use bulk liquid culture combined with genetic or drug-induced blocking of progeny, introducing physiological stress on the animals. Common small-scale pilot screens adopt agar-based methods, which necessitate the tedious task of repetitively picking and transferring animals. In both approaches, it is challenging to add or remove reagents (e.g., food or drugs) at multiple time points during the lifespan, impairing detailed aging investigations. We report a compact and modular lifespan machine based on microfluidics that addresses the limitations of current methods. Our lifespan machine is comprised of three modules, (i) a microfluidic lifespan chip, (ii) a fluid exchange module and (iii) an imaging module. The microfluidic device houses crawling animals and an on-chip filter that allows the selective removal of progeny. The fluid exchange module controls the flow rate, duration and frequency of fluid injection for washing out progeny and for regularly feeding worms. A microcontroller is integrated into the system that is commanded by a simple GUI, allowing user-defined control of the fluid exchange module. The imaging module consists of a camera sensor and on-board Wi-Fi that captures, transmits and stores images at user-defined intervals. We designed custom software that analyzes the locomotory motion and conducts lifespan analysis. The entire system has a footprint of no more than 20 x 20 cm2 and has been designed such that it does not require any physical interaction, once the lifespan experiment is initiated. To reduce physiological stress on the animals, we have optimized the device geometry and feeding protocols such that C. elegans gait, body size, and lifespan are consistent with aging assays on agar. We find that the lifespan curves of wild-type and genetic mutants are consistent with the literature reports. In summary, the compactness and modularity of our approach allows building several of these systems, enabling highly parallelized cross-sectional and longitudinal aging experiments.

Powell-Coffman JA et al. (1998) Midwest Worm Meeting "Characterization of ahr-1 and aha-1: the C. elegans AHR signaling complex"

Cary CL, Jiang H, Powell-Coffman JA, Wood WB

[

Midwest Worm Meeting,

1998]

ahr-1 and aha-1 are the orthologs of the mammalian aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT), respectively. AHR is a ligand-activated transcription factor, which mediates the carcinogenic and teratogenic effects of certain environmental contaminants. ARNT is the only transcription factor known to dimerize with AHR, but ARNT can dimerize with several partners in vitro, including Drosophila Single-minded and Trachealess. Endogenous ligands for AHR have not been identified, and the cellular and developmental functions of AHR are not understood. We are investigating the role of ahr-1 and aha-1 in C. elegans development, and we will address outstanding questions concerning the function and regulation of the aryl hydrocarbon receptor signaling complex. In collaboration with Chris Bradfield (Madison, WI), we determined that AHR-1 and AHA-1 share important biochemical properties with their mammalian cognates. Specifically, AHR-1 forms a tight association with HSP90, and AHR-1 and AHA-1 interact to bind DNA fragments containing the mammalian xenobiotic response element with sequence specificity. Yeast expression studies indicate that C. elegans AHR-1, like vertebrate AHR, requires some form of post-translational activation. Moreover, this requirement is dependent on the presence of the domains predicted to mediate binding of HSP90 and ligand. Preliminary experiments suggest that the spectrum of AHR-1 agonists is different from that of the mammalian receptor, and we are using a yeast assay system to test candidate AHR-1 ligands. Strains containing Tc1 transposon insertions in aha-1 and ahr-1 were isolated from libraries constructed in the laboratories of Jim Priess and Joe Culotti, respectively. We are screening for deletions induced by Tc1 excision, and we have identified two deletions in aha-1 that are transmitted through the germline. Allele ia01 is a 2454 bp deletion of aha-1, and it is predicted to encode only 54 amino acids, including half of the DNA-binding domain. We will present preliminary molecular and phenotypic analyses of the mutations at the meeting. We are also studying the cellular and subcellular localization of these proteins. Most cells that express AHR-1::GFP also express AHA-1::GFP. These include some chemosensory neurons and intestinal cells. Additionally, AHA-1::GFP is expressed in many neurons and pharyngeal cells that do not express the receptor. We have also generated polyclonal antisera that recognize AHA-1, and we are purifying AHA-1-specific antibodies.

Juan Cueva et al. (2007) International Worm Meeting "The Nanoscale Relationship Between the MEC-4 Transduction Channel and the Extracellular Matrix Molecule, MEC-5."

Miriam Goodman, Juan Cueva

[

International Worm Meeting,

2007]

Mechanotransduction is the first step in hearing, pain, and touch. In C. elegans, displacement of the body wall opens MEC-4/degenerin transduction channels in touch receptor neurons (TRN). Touch sensation also requires components of a characteristic 15-protofilament (15-pf) microtubule bundle and extracellular matrix (ECM). Genetic interactions between the genes encoding the MEC-4 channel, 15-pf microtubules, and ECM led to the hypothesis that MEC-4 channel gating occurs in a tethered mode. Previously, we used novel antibodies against the MEC-4 channel subunits, MEC-2 and MEC-4, to establish that the MEC-4 channel is present intracellularly on the 15-pf microtubules and on the plasma membrane.1 The membrane-associated MEC-4 channels are distributed around the circumference of the TRNs and separated by >100 nm from the nearest points of contact between the 15-pf microtubules and the plasma membrane. Thus, the MEC-4 channels are not consistently associated with the 15-pf microtubules and unlikely to be tethered by them. However, the spatial relationship between membrane-associated MEC-4 channels and the ECM remains unclear. We tested the hypothesis that the MEC-4 channel is associated with the ECM molecule MEC-5 using post-embedding immunoelectron microscopy. First, we developed antibodies against the collagen-like molecule MEC-5. At the light level, MEC-5 antibodies label a single row of uniform puncta on the TRN. On average, MEC-5 puncta are 0.27 ± 0.07 mm (n = 142 puncta) long or less than half the size of the puncta labeled by antibodies against MEC-2 (0.75 ± 0.36 mm, n = 625 puncta) and MEC-4 (0.74 ± 0.32 mm, n = 399 puncta). In addition, the MEC-5 puncta are more numerous and more dense than either the MEC-2 or MEC-4 puncta. The labeling with the MEC-5 antibodies is specific since TRN neurites were not labeled in mec-5 null mutants. Second, we used the MEC-5 antibodies to label TRNs in 50 nm thin sections. MEC-5 antibodies were detected within the specialized electron-dense ECM between the TRN neurites and the surrounding hypodermal cell. Consistent with published genetic and molecular observations, the MEC-5 label, like the electron-dense ECM, was concentrated at the apical (cuticle) side of the neurite. These data argue that the MEC-4 channels and MEC-5 are co-expressed in the TRN neurite but may not interact physically. Thus, previously reported genetic interactions between mec-5 and mec-7 b-tubulin are unlikely to be mediated by a common complex incorporating MEC-5, the MEC-4 transduction channel, and the 15-pf microtubules. 1Cueva & Goodman, (2006) Neuronal Development, Synaptic Function, and Behavior, Topic Meeting #2, Madison, WI. Online.