Nie, Yu et al. (2013) International Worm Meeting "Oligomeric proanthocyanidins extracts are putative anti-obesity targets in the nematode Caenorhabditis elegans."

Sturzenbaum, Stephen, Hider, Bob, HYLANDS, PETER, Nie, Yu, Bansal, Sukhi

[International Worm Meeting, 2013]

Proanthocyanidins are among the most common secondary metabolites in plants and thus are crucial to human diet and therefore health. Oligomeric proanthocyanidins (OPCs, DP 5) are thought to be effective in improving lipid homeostasis in rodent animals and humans, which may be attributed to their good bioavailability. The effectiveness of OPCs as an anti-obesity treatment has been tested but poorly described due to the vague understanding of their composition. Besides, an in vivo study using purified OPCs is yet to be elucidated, a shortfall we aim to address here. Owing to the rapid screen and high-throughput capacity, Caenorhabditis elegans is well positioned to advance the knowledge of OPCs' mode of action and efficiency as an anti-obesity drug. Purification of OPCs extracts from grape seeds was realized on preparative-thin layer chromatography (TLC) coupled with reverse-phase high performance liquid chromatography (RP-HPLC), confirmed by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy(NMR). The growth of age-synchronized N2 worms exposed to OPCs was measured. Lipid stores and lysosomal compartments within the worm bodies were visualized by staining with the hydrophobic fluorescent dye Nile Red and LysoTracker. In parallel, a biochemical assay was applied to quantify the triglyceride level. Statistical significance testing was performed with Prism GraphPad5. In an attempt to purify crude extracts, pure monomers were collected following a RP-HPLC and verified by spectroscopy. Prep-TLC yielded pure dimers, trimers and tetramers confirmed by MS. Worms exposed to OPCs decreased in size, an effect that was most significant with trimer- to pentamer-enriched OPC fractions. A statistically significant decrease was observed in Nile Red staining. Similarly, triglyceride level decreased most when worms were treated with dimer- and trimer-enriched fractions. Exposure to OPC enriched fractions decrease the volumetric surface area and lipid storage capacity of C. elegans, confirming their potential as anti-obesity leads. OPCs trimers are predicted to be the most effective.

Kornfeld K et al. (2000) Midwest Worm Meeting "Molecular and Genetic Analysis of lin-1 mutations."

Kelly GR, Kornfeld K

[Midwest Worm Meeting, 2000]

The formation of the C. elegans hermaphrodite vulva is controlled by multiple signaling pathways. The highly conserved RAS pathway promotes the primary vulval cell fate by negatively regulating the ETS transcription factor LIN-1. Bob Horvitz and colleagues isolated 49 alleles of lin-1 in a variety of screens for vulval defects. These alleles include loss-of-function and gain-of-function mutations of different severity. The molecular lesions responsible for some alleles have been identified, but many have yet to be characterized. To identify residues and domains that are important for LIN-1 structure and function, we are using DNA sequencing to determine the molecular lesions of these alleles.

Theresa Stiernagle et al. (2007) International Worm Meeting "Caenorhabditis Genetics Center."

Robert K Herman, Theresa Stiernagle, Ann E Rougvie

[International Worm Meeting, 2007]

The Caenorhabditis Genetics Center (CGC), supported by NIH NCRR, supplies Caenorhabditis strains and information to researchers throughout the world. On May 31, 2007, which marks the end of our current 15-year contract, Ann Rougvie will replace Bob Herman as CGC Director, and Bob will retain an advisory role as a Professor Emeritus. The CGC will continue to be housed at Minnesota and curated by Theresa Stiernagle. A five year renewal contract was submitted in August and is pending at NIH. We anticipate that a final (and positive) funding decision will be in hand by the start of the 2007 worm meeting. The CGC will continue its duties of acquiring, maintaining and distributing worm stocks, but other past activities of the CGC have been subsumed by WormBase. These include generating and maintaining a C. elegans bibliography, providing a clearinghouse for C. elegans genetic nomenclature, and maintaining a genetic map. The CGC now has over 8550 different strains. We strive to have at least one allele of every published gene and all chromosome rearrangements, duplications and deficiencies. In addition, we have several strains of species closely related to C. elegans. A searchable strains list will continue to be accessible either through the CGC website (www.cbs.umn.edu/CGC/) or through WormBase. Strains are available upon written request, which should include a brief statement of the intended use of the strains. Email requests (to cgc@umn.edu) are satisfactory. We provide quarterly reports to the NIH with statistics that reflect our services to the worm community. We like to be acknowledged in papers for providing strains. We also like to receive reprints of worm papers (.pdf files are encouraged), copies of which we also provide to NIH.

BD Galvin et al. (1999) International C. elegans Meeting "Genetic and molecular analyses of ced-8, which controls the time of appearance of cell corpses"

HR Horvitz, BD Galvin

[International C. elegans Meeting, 1999]

In C. elegans development, the timing of programmed cell deaths in the soma is essentially invariant. The molecular mechanisms that control this timing are poorly understood. Mutations in ced-8 result in the delayed appearance of cell corpses. These mutations were originally identified in screens for mutations that cause the presence of cell corpses in late-stage embryos. ced-8 has been cloned (G.Stanfield, M. Hengartner, and Bob Horvitz; 1995 International Worm Meeting abstract 486) and appears to encode a multiple-pass transmembrane protein. To determine the role that ced-8 plays in programmed cell death, we intend to use both genetic and molecular approaches. We will screen for suppressors of the ced-8 phenotype. Specifically, using a sem-4; ced-8 mutant background, we will screen for F2 bags that contain F3 embryos wild-type in the timing of cell deaths. This screen may elucidate the role of ced-8 in programmed cell death and identify additional genes involved in cell-death execution. We also will generate antibodies for immunohistochemistry and perform biochemical assays to address the function of CED-8.

JJ Yochem (1999) International C. elegans Meeting "Mutations that confer aspects of the molting defects seen with lrp-1 mutations or sterol starvation"

JJ Yochem

[International C. elegans Meeting, 1999]

Mutations that produce larvae that are completely stuck in old cuticle, that have rings of unshed cuticle that constrict regions of the body, or that have attached to the tail a train of old cuticle will be described. Such larvae can be detected with a dissecting microscope, permitting a semi-clonal screen. The search for such mutations was begun because a failure to complete molting is an aspect of the phenotype conferred by loss-of-function mutations in lrp-1 , the product of which resembles mammalian gp330/megalin, a large member of the LDL receptor family of proteins [Yochem et al., Development 126, 597 (1999)]. It is hoped that new mutations will reveal genes whose products are required for the same process as LRP-1. The mutations also have the potential for increasing our understanding of molting, a process that has not been much studied genetically in nematodes. To date, mutations have defined eight candidate genes. Of particular note are mlt-3(sv8) and mlt-7(sv16) , both of which closely resemble the lrp-1 mutant phenotype. Thanks to Simon Tuck, Min Han, and Bob Herman.

Alfonso A et al. (1995) International C. elegans Meeting "MOLECULAR ANALYSIS OF THE GENE UNC-11."

Hollenback CE, Alfonso A, McCarron A

[International C. elegans Meeting, 1995]

Our long term goal is to understand the molecular and genetic bases of regulated neurotransmitter vesicular release. To pursue this goal we are characterizing the structure and function of the gene unc-11. Collaborative studies of several laboratories, including ours, have indicated that unc-11 mutants are defective in the release of two neurotransmitters, acetylcholine and gamma amino butyric acid. These studies strongly implicate a function in synaptic neurotransmitter vesicular release. We have identified the genomic region comprising the gene unc-11 by the technique of DNA-mediated transformation rescue. We have found that the genomic cosmids C32E8 and C05F5 are necessary and sufficient to rescue the unc-11 mutant phenotypes. We have subcloned the genomic sequences comprising most of the gene and used them to screen a cDNA library obtained from Bob Barstead. We have characterized four cDNAs, all are shorter than the RNA detected in Northern blot analysis. The cDNAs have been characterized by restriction mapping and DNA sequencing; all four share sequences at the 3'end but differ at their 5' ends. The differences at the 5'end appear to result from alternative splicing. We will present our findings and the deduced protein sequence.

Park M et al. (1996) East Coast Worm Meeting "IDENTIFYING FACTORS THAT INTERACT WITH amide IN BODY WALL MUSCLE DEVELOPMENT"

Littlejohn R, Krause MW, Park M

[East Coast Worm Meeting, 1996]

The MyoD family of transcription factors are involved in the specification and development of vertebrate skeletal muscle. These factors contain a conserved basic helix-loop-helix (bHLH) domain that directs DNA binding and protein dimerization. In vertebrates, the MyoD family members heterodimerize with an ubiquitously expressed E protein to activate muscle specific expression. A single homolog of MyoD (CeMyoD) and E proteins (CeE/DA) has been found in C. elegans . Although CeMyoD is expressed in the nuclei of all body wall muscle cells and their precursor blastomeres, CeE/DA is not expressed in these cells. This result raises the question of whether there are other factors that heterodimerize with CeMyoD to direct proper body wall muscle development. To address this question, we have utilized the yeast two-hybrid system to identify gene products that interact with CeMyoD. We have used a construct containing the bHLH domain of CeMyoD fused to the DNA binding domain of Gal4 to screen a cDNA library containing fusions to the Gal4 activation domain (kindly provided by Bob Barstead). In a screen of 700,000 clones, we identified 3 clones that interact with the CeMyoD bHLH-Gal4 BD fusion. We will report on the characterization of these interacting clones.

Wissmann AK et al. (1995) International C. elegans Meeting "LET-502 IS A HOMOLOG OF HUMAN MYOTONIC DYSTROPHY KINASE"

Wissmann AK, Mains PE, McGhee JD

[International C. elegans Meeting, 1995]

We have isolated a mutation (ca201) in the let-502 gene which leads to embryonic arrest at early morphogenesis. Ann Rose's lab had originally identified 5 other mutations in let-502 that arrest between morphogenesis and adult-hood. We have shown that all 6 alleles are likely dominant-negatives and that let-502 is expressed maternally and zygotically. The ca201 allele was rescued by cosmid injections. cDNA's were isolated from Bob Barsteadt's cDNA library and the largest (4.3kbp) was sequenced. Sequence analysis suggests that LET-502 is a homolog of human myotonic dystrophy kinase. The N-terminal regions (400aa) of both show strong homology and include a cAMP-dependent serine/threonine kinase domain. These regions are followed in both by a coiled-coil motif. Preliminary results with lacZ fusion constructs in adults suggest expression in pharyngeal muscle cells, several cells located in the vicinity of the vulva and a few cells in the tail. In the embryo we have tentatively identified staining in at least some body wall muscle cells. Myotonic dystrophy in humans is due to triplet expansion in the 3'-untranslated coding region. The actual function of the kinase remains unclear. Analysis of the C.elegans mutants and the use of supression genetics should facilitate our understanding of myotonic dystrophy.

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

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

[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.

Ginger Kelly et al. (2001) International C. elegans Meeting "Analysis of lin-1 Mutants and Screen for Suppressors of lin-1(n383)"

Ginger Kelly, Kerry Kornfeld

[International C. elegans Meeting, 2001]

A highly conserved RTK/Ras/MAP kinase pathway promotes the primary vulval cell fate by negatively regulating the ETS transcription factor LIN-1. Bob Horvitz and colleagues isolated 41 alleles of lin-1 in a variety of screens for vulval defects. These alleles include loss-of-function and gain-of-function mutations of different severity. To identify residues and domains that are important for LIN-1 structure and function, we used DNA sequencing to determine the molecular lesions of these alleles. The loss-of-function mutations include several missense mutations that affect the ETS domain and a series of nonsense mutations. The DNA-binding properties of the ETS domain mutants are being investigated. To identify genes that function downstream or in parallel to lin-1 to promote the primary cell fate, we are screening for mutations that suppress the multivulva (Muv) lin-1(n383) loss-of-function phenotype. We have screened about 5,500 haploid genomes and identified ten suppressors of lin-1 Muv. These ten mutations cause sterility. These suppressor mutations will being mapped relative to polymorphisms between the N2 strain and the Hawaiian CB 4856 strain. lin-1 is epistatic to the well characterized genes in the RTK/Ras/MAP kinase pathway, therefore these mutations may affect new genes that function downstream of lin-1 to regulate the primary cell fate.