Brockett, Mary et al. (2015) International Worm Meeting "Virulence of a Novel Melanin Producing Pseudomonas Species UC17F4 on Caenorhabditis elegans."

Brockett, Mary, Morreale, Meghan, Aaronson, Lawrence, Shinn-Thomas, Jessica

[International Worm Meeting, 2015]

Pseudomonas sp. UC17F4 is a novel species of Pseudomonas isolated in our lab from the cutaneous microbial flora of the red-backed salamander, Plethodon cinereus. UC17F4 produces intracellular melanin, a brown pigment, in the presence of tyrosine-rich media. Melanin has been shown to serve as a virulence factor for several organisms, including bacteria and fungi. Our lab generated several mutant strains of UC17F4: UC17F4 (MM1) produces less melanin than the wild-type strain, UC17F4 (MM7, 8, and 9) hypersecrete extracellular melanin, and UC17F4 (PV 21 and 22) produce no melanin. We demonstrated that UC17F4 exhibits virulence by using C. elegans as a model host. Worms were transferred to lawns of UC17F4 bacteria on Nematode Growth Media (NGM) supplemented with tyrosine and lethality was assessed over time using touch assays. Our studies show that C. elegans exposed to the wild-type strain (UC17F4) have the highest mortality rate and worms exposed to UC17F4 (PV21) have the lowest mortality. Worms do not die after exposure to the hypersecreting strains or the strains without melanin. We investigated the effect of UC17F4 exposure on the different larval stages of C. elegans (L1, L2, L3, L4, and adult worms). Our results show that L1 and L2 worms are most vulnerable to the virulence of UC17F4 compared to later developmental stages. L1 and L2 worms die in less than 24 hours of exposure, whereas later developmental stages are viable and reproduce. Our current studies include determining the kinetics of L1 and L2 lethality using both touch assays and in vivo fluorescent cell death assays (SYTOX®) in a microplate reader, and determining the mode of pathogenesis using high-magnification light microscopy to examine the anatomical areas of melanin and UC17F4 biofilm accumulation. .

Keith A Boroevich et al. (2004) West Coast Worm Meeting "Identification of Regulatory Elements in Caenorhabditis elegans using Orthology Biased Gibbs Sampling"

Steven JM Jones, David L Baillie, Keith A Boroevich

[West Coast Worm Meeting, 2004]

Much attention has recently been placed on the problem of identification of gene regulatory sequences. Two main computational approaches exist: 1) identification of sequences that share similarity to known regulatory elements, and 2) de novo identification of common motifs in a set of co-regulated genes. Orthology Biased Gibbs Sampling (OrBS) applies a modification of the Gibbs Sampler put forth by Lawrence, Altschult et al . 1 in a attempt to solve this problem. Additional features include analysis of negative strand, multiple (or no) occurrences of a motif in any given sequence and identification of multiple unique motifs, all of which have appeared in previous incarnations of the Gibbs Sampler. The unique feature of OrBS is the use of comparative genomics as an informative prior and in the calculation of motif probability. OrBS will be applied to the identification of regulatory elements C. elegans . Clustering of spatially co-regulated genes as defined by the C. elegans Gene Expression Project (see Johnsen et al . poster). This data is more amenable to regulatory element detection than the more common microarray data because the sequence in which a cis -acting element may reside is strictly defined. Interspecies sequence conservation will be identified through alignment of the C. elegans gene promoter region to the promoter region of the C. briggsae ortholog using LAGAN 2 . Predicted regulatory elements will be verified in vivo using site directed mutagenesis and promoter::GFP fusions. References: 1 Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. (1993). Science 262: 208-214. 2 Brudno M, Do C, Cooper G, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). Genome Research 13: 721-731.

Mavji N Patel et al. (2001) International C. elegans Meeting "Evolution of germ-line signals that regulate growth and ageing in nematodes"

Armand M Leroi, Constantina Karageorgi, Christopher G Knight, Mavji N Patel

[International C. elegans Meeting, 2001]

One of the least understood aspects of animal development is how growth and body size are regulated 1,2 . Here we show that a signal from the germ-line represses growth in the nematode Caenorhabditis elegans . Laser-microbeam ablation of cells that give rise to the germ-line causes adults to become giant. Ablation of these cells in self-sterile mutant worms also causes gigantism suggesting that the germ-line represses growth because it is the source of a growth-antagonizing signal rather than a sink of resources required for reproduction. The C. elegans germ-line also emits a signal that represses longevity 3 . This longevity-repressing signal requires the activity of DAF-16, a forkhead/winged-helix transcription factor 3 , but we find that that the growth-repressing signal does not. The growth-repressing signal also does not require the activity of DBL-1, a TGF-beta-related protein that promotes growth in worms 4,5 . By ablating the germ-line precursors of other species of free-living nematodes we found that both the growth-repressing and longevity-repressing signals are evolutionarily variable. Some species have both, others have just one or the other. We suggest that variation in germ-line signaling contributes to body size and life-history diversity in the nematodes. 1. I, Conlon, M. Raff, Cell 96, 235 (1999). 2. S. J. Day, P. A. Lawrence, Development 127, 2977 (2000). 3. H. Hsin, C. Kenyon, Nature 399 , 362 (1999). 4. K. Morita, K. L. Chow, N. Ueno, Development 126, 1337 (1999). 5. Y. Suzuki et al ., Development 126, 241 (1999).

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.

Donavan TS Cheng et al. (2007) International Worm Meeting "A NON-ALIGNMENT GIBBS-SAMPLING BASED METHOD FOR SEQUENCE ELEMENT DETECTION USING PHYLOGENETIC CONSERVATION."

Donavan TS Cheng, Michael A Beer

[International Worm Meeting, 2007]

Current attempts to infer regulatory networks from genome-wide expression data require motif detection algorithms to identify transcription factor (TF) binding sites in the regulatory regions of co-expressed genes. These algorithms typically use either over-represention or phylogenetic conservation to predict TF binding sites. While these approaches have been successful in yeast [1,2,5], in larger genomes motif detection is significantly more challenging, and will likely require comparative genomics. Several recent algorithms have begun to combine these two sources of information to predict functional sites more accurately. [1,2] However, many rely on an initial global alignment of orthologous regulatory regions as a preprocessing step. Unfortunately, TFs generally recognize sites that are short and degenerate, and global alignment may be overly restrictive for the detection of such sites. We have developed a non-alignment based Gibbs sampling [3,4] algorithm that searches for over-represented sequence elements, conserved across orthologous regulatory regions in related species. Motif composition is represented probabilistically by a position weight matrix and a posterior probability score is used to assess the significance of the motif found. The algorithm employs a stochastic search to converge to optima and unlike other alignment-based techniques, does not require a prior estimate of the evolutionary relationship between species sampled. We compare the performance of our algorithm on synthetic data to that of existing algorithms that employ either over-representation, phylogenetic conservation, or both. We further validate our motif predictions on in vivo binding data in yeast. [5] We present novel motif predictions from sets of coexpressed genes C. elegans and the related sequenced nematode species C. briggsae, and C. remanei. 1. Siddharthan R, Siggia ED, van Nimwegen E. PLOS Comp. Biol. 1, e67 (2005). 2. Sinha S, Blanchette M, Tompa M. BMC Bioinf. 5, 170 (2004). 3. Roth FP, Hughes JD, Estep PW, Church, GM. Nat. Biotech. 16, 939-945 (1998). 4. Neuwald AF, Liu JS, Lawrence CE. Prot. Sci. 4, 1618-1632 (1995). 5. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, et al. Nature 431, 99-104 (2004).

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.

Elizabeth A De Stasio (2003) International Worm Meeting "Using C. elegans in a Molecular Biology Laboratory Course"

Elizabeth A De Stasio

[International Worm Meeting, 2003]

Molecular Biology at Lawrence enrolls 35-45 undergraduates annually and includes three lectures and one three-hour lab/discussion section per week. Students have access to the lab and are expected to complete lab work on their own.Module 1, PCR: In previous years, students used PCR to identify deletions in genes unc-93 or unc-54. Students were given primer sets known to identify deletions in particular strains as well as plates of wild type and mutant animals. Students did a crude DNA extraction, set up the reactions, and analyzed PCR products using an acrylamide gel. This year Ive included site-directed mutagenesis. Student lab groups were given a computer file containing the unc-54 gene sequence and told to design a particular kind of mutagenic primer and an upstream primer covering a restriction site. Students used computer programs to test the binding specificity of their primers and to predict the product size. Each lab section settled on one primer design. Over the course of two weeks, students prepared PCRs, digested PCR products as well as a plasmid containing unc-54 with the chosen restriction enzymes, and purified the products from a low-melt agarose gel. The module concluded with production of ligation reactions which were handed to a research student for further analysis. If a submitted NSF grant proposal is funded, this laboratory exercise will continue for a third week next year when students will analyze their putative mutant constructs using an automated DNA sequencer.Module 2, Reporter Genes: In this qualitative, easy lab students learn the value of reporter gene constructs for analysis of gene expression patterns. It is a good lab to schedule at the end of a spring course when seniors dont want to work hard. Students are provided with two different mixed populations of worms, identified by number, each containing an lac-Z::promter fusion as an integrated transgenic array. Students permeabilize the worms and stain for B-galactosidase activity. Students carefully observe staining of different tissues in different ages of worms and draw examples of their observations before being told which promoter fusion they had. Students then use available databases to confirm their observations. Promoters used successfully thus far include myo-2, unc-54, and pes-10 (the generous gift of Bill Morgan, College of Wooster). The use of lac-Z rather than GFP allows simultaneous observations by many students using simple dissecting microscopes. I would be happy to receive other strains containing other lac-Z fusions!

Lawrence Schriefer et al. (2001) International C. elegans Meeting "Defining Regulatory Regions In Nematode Muscle Genes By Computational And Transgenic Studies."

Robert Waterston, Gary Stormo, Lawrence Schriefer, Debraj GuhaThakurta, Michelle Hresko

[International C. elegans Meeting, 2001]

We have initiated experiments designed to understand the regulatory regions of C. elegans genes using known muscle genes of C. elegans as a model. Two approaches are being used to pursue this goal. The first approach is to computationally compare the muscle genes from C. elegans to the orthologous muscle sequences from C. briggsae . This comparison is useful because the patterns of gene regulation and regulatory elements are often conserved across species. The C. briggsae orthologue are found by making a probe from the C. elegans muscle gene and probing the C. briggsae fosmid filter available from Incyte. The most promising positive clones are determined by fingerprinting and these are sequenced by the Genome Sequencing Center. To compare the orthologous sequences from C. elegans and C. briggsae , we will use pairwise alignment methods like BlastZ(4) or Bayes aligner(5) to identify regions of interest. Local multiple alignment programs can then be used to search for common regulatory elements in these regions. Since the local multiple alignment methods work best with sequences which are only 1000-2000 nucleotides long, phylogenetic footprinting will be useful in identifying shorter regions from much longer regions(10,000-20,000 nucleotides). The second approach is to use a combination of computational methods to identify potential muscle specific regulatory elements from the known set of C. elegans muscle genes. Local multiple sequence alignment methods like Consensus(1), Ann-Spec(2) and Co-Bind(3) are being used to identify these potential regulatory elements. Using the above method we have already identified several potential regulatory elements which show high degree of specificity for the muscle genes. The regulatory elements that these computational methods predict can then be used to screen the C. elegans genome for new genes that are expressed in muscle cells. To test our results we have developed a method to examine the expression patterns of genes in C. elegans using gfp promoter fusions. We are including in our promoter fusions 6,000 nucleotides upstream of the start methionine, all of the first exon and all the first intron. In our initial experiments, known muscle genes tested in this manner show muscle-like expression. We can now use this method to test the requirement for regulatory regions predicted by the computational work to determine if they convey muscle specific expression. In addition, we can use this method to test genes we predict to be, but not previously known to be, expressed in muscle. Furthermore, we are developing these methods to allow for the rapid production of these promoter fusions so that ultimately, a genome wide program to categorize all C. elegans genes by gfp and automated lineaging can be done. 1. Hertz, G.Z., and Stormo, G.D. (1999) Bioinformatics, vol. 15, pp. 563-577 2. Workman, C.T., and Stormo, G.D. (2000) Pacific Symposium on Biocomputing, vol 5, pp. 464-475 3. GuhaThakurta, D., and Stormo, G.D. (2001) Bioinformatics, in press. 4. Schwartz, S. et.al. (2000) Genome Research, vol. 10, pp. 577-586. 5. Zhu, J., Liu, J.S., and Lawrence, C.E. (1998) vol. 14, pp. 25-39.