Simske JS et al. (1994) Worm Breeder's Gazette "Specification of Vulval Cell Fates By Sequential Signalling Pathways."

Simske JS, Kim SK

[Worm Breeder's Gazette, 1994]

Specification of vulval cell fates by sequential signaling pathways Jeffrey S. Simske and Stuart K. Kim, Dept. of Developmental Biology, Stanford University Medical Center, Stanford CA 94305

HE Smith et al. (1999) International C. elegans Meeting "Genome-wide analysis of C. elegans sperm development"

S Ward, JF Nance, HE Smith, EB Davis

[International C. elegans Meeting, 1999]

Our lab is interested in the developmental processes that direct sperm formation in Caenorhabditis elegans . Prior genetic screens have identified 60 genes required for proper sperm development, and several of these fer (fertilization-defective) or spe (spermatogenesis-defective) genes have been cloned by cosmid rescue. However, analyzing the function of these genes has been hampered by the length of time required to clone each gene, and the lack of homology to genes of known function. Because all of the previously cloned fer/spe genes exhibit sperm-specific expression, the identification of those genes expressed only in the developing sperm would speed cloning and identify a subset of genes with homologs. The recent completion of the genome sequence offers the opportunity for genetic analysis on a global scale. Therefore, we are attempting to identify all of the estimated 400 genes expressed solely during sperm development by 1) sequencing clones from a sperm-specific subtracted cDNA library and 2) differential screening of microarrays (in collaboration with the laboratory of Stuart Kim). Both strategies utilize temperature-sensitive mutant hermaphrodites that produce only oocytes ( fem-1 ) or sperm ( fem-3gf ) at the restrictive temperature. Because the somatic tissues are unaffected, any difference in gene expression between these two mutants is due to differences between sperm and oocyte production. We have constructed two high-quality cDNA libraries from mRNA isolated from fem-1 and fem-3gf mutants. Both libraries have been normalized to equalize the relative representation of abundant vs. rare transcripts. The fem-1 library has been subtracted from the fem-3gf library to generate a library enriched to ~20% sperm-specific genes. Reverse Northern blots with fem-1 and fem-3gf probes have been used to identify sperm-specific clones, which are being sequenced by the Genome Sequencing Center at Washington University. By comparing these results to Stuart Kim's differential microarray screening with fem-1 and fem-3gf probes, we can assay the sensitivity and specificity of these two methods. We will also present data from our current attempts to improve germline expression of transgenic arrays as well as increased efficacy of germline RNA inactivation.

Pilgrim DB et al. (1991) International C. elegans Meeting "MOLECULAR ANALYSIS OF THE DAUER CONSTITUTIVE daf-7 GENE."

Johnsen RC, Pilgrim DB, Riddle DL

[International C. elegans Meeting, 1991]

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tcl tagging. On a chromosomal walk toward fem-2, one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9, which contains an insert of <30kb, was used to probe both a northern blot and a CDNA library constructed by Stuart Kim. The northern blot revealed a single hybridizing RNA, and ten cDNA clones of various sizes up to 2.4kb were isolated from the library. Hybridization results indicate that all these clones share common sequences. Probing restriction digested genomic DNA isolated from the Tcl-insertion mutant DR1039, daf-7 (m434) , revealed differences consistent with a 1.6kb Tcl insertion in DR1039 DNA when compared with nonmutant DNA. For example, a 2.2kb EcoRl fragment found only in mutant DNA hybridized to both DE9 and to Tcl. We intend to determine the sequence of a daf-7 cDNA in order to obtain information regarding its possible function, and possible relationship to the daf-l and daf-4 receptor protein kinases.

Beitel GJ et al. (1991) International C. elegans Meeting "CLONING AND SEQUENCING OF THE SYNTHETIC MULTIVULVAL GENE lin-9"

Beitel GJ, Horvitz HR

[International C. elegans Meeting, 1991]

A synthetic Multivulva phenotype can result from mutations in two separate genes, while each mutation alone results in an apparently wild-type phenotype (Ferguson and Horvitz, Genetics 123:109-121,1989). Mutations that can cause this synthetic Multivulva (syn Muv) phenotype have been grouped into two classes, A and B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. Iin-9(nll2) III is a class B mutation that results in an apparently wild-type phenotype by itself. We previously reported that ZK637 and overlapping cosmids can rescue lin-9 (WBG 11(2 ): p 29,1990). We have now identified an 8 kb subclone with rescuing activity. Using this 8 kb fragment to probe Stuart Kim's cDNA library, six positive plaques representing at least three independent clones were isolated. This 8 kb probe also detects a message of approximately 2.5 kb on Northern blots. The message level appears similar in eggs and mixed-stage populations. We are currently sequencing these cDNAs and looking for evidence that these transcripts do in fact represent the lin-9 gene. This work will be greatly aided by the genomic sequence provided by Molly Craxton, John Sulston and Alan Coulson, as ZK637 was fortuitously chosen as one of the first cosmids to be sequenced in the worm genome sequencing project.

Pilgrim DB et al. (1992) Worm Breeder's Gazette "Preliminary Sequence Analysis of the daf-7 Gene"

Johnsen RC, Riddle DL, Pilgrim DB

[Worm Breeder's Gazette, 1992]

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tc1 tagging. On a chromosomal walk toward fem-2 ,one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9 was used to probe a cDNA library constructed by Stuart Kim. Twenty cDNA clones of various sizes up to 2.3kb were isolated from the library. Hybridization results indicate that all these clones share common sequences. Probing restriction digested genomic DNA isolated from the Tc1 -insertionmutant DR1039 ,daf-7 (m434),revealed differences consistent with a 1.6kb Tc1 insertion in DR1039 DNA when compared with non-mutant DNA. For example, when probed with cDNA a 11kb BamHI fragment found in wild-type genomic DNA corresponds to a 12.6kb fragment in mutant DNA. We sequenced both ends of the largest (2.3kb) cDNA clone, and have orfs totaling 460 amino acids. Unfortunately, no appropriate initiator codon or poly A addition site are evident in the cDNA. Therefore, we are screening Bob Barstad's cDNA library to identify a more complete clone. The sequence so far shows no strong homology to anything in the NBRF-PIR data base, although it does have weak homology to some calcium dependent ATPases.

Steven M. Johnson et al. (2007) International Worm Meeting "Toward a high-resolution nucleosome position map of the Caenorhabditis elegans genome."

Frederick J. Tan, Arend Sidow, Thaisan Tonthat, Heather L. McCullough, Anton Valouev, Kathy Zeng, Heather Peckham, Gina Costa, Andrew Fire, Daniel P. Riordan, Steven M. Johnson, Joel Malek, Jeremy Stuart, Kevin McKernan, Jeffrey Ichikawa, Swati Ranade

[International Worm Meeting, 2007]

We are working toward a detailed structural and dynamic picture of C. elegans chromatin. Nucleosome positions within the chromatin landscape are known to serve as a major determinant of DNA accessibility to transcription factors and other interacting components. To delineate nucleosomal patterns in C. elegans, we are carrying out a genome-wide analysis in which DNA fragments corresponding to nucleosome cores are liberated using Micrococcal nuclease. Sequence analysis of an initial set of putative nucleosome cores obtained in this manner from a mixed-stage population of C. elegans reveals a combined picture of flexibility and constraint in nucleosome positioning. As had previously been observed in studies of individual loci in diverse biological systems, we observe areas in the genome where nucleosomes can adopt a wide variety of positions in a given region, areas with little or no nucleosome coverage, and areas where nucleosomes reproducibly adopt a specific positional pattern. In addition to illuminating numerous aspects of chromatin structure for C. elegans, this analysis provides a reference from which to begin an investigation of relationships between the nucleosomal pattern, chromosomal architecture, and lineage-based gene activity on a genome-wide scale. We are currently extending this analysis using ultra-high-throughput sequencing techniques analyzing the genomic positions of millions of nucleosome cores toward the end of producing a high-resolution nucleosome position map of the C. elegans genome.

Monica C Sleumer et al. (2005) International Worm Meeting "The Search for Regulatory Elements in C. elegans"

Obi L Griffith, Steven JM Jones, Monica C Sleumer, Xin Zhang, A Gordon Robertson, Mikhail Bilenky, Erin D Pleasance

[International Worm Meeting, 2005]

The availability of large amounts of genomic and expression data provides an excellent opportunity to examine the relationship between gene expression, orthologous genes and known intergenic regulatory elements to search for novel functional regulatory elements in C. elegans on a genome-wide scale. For each gene in C. elegans, a list of putative coregulated genes consisting of coexpressed genes and orthologues from other nematode species is assembled. The coexpressed genes are obtained from available expression data, such as the cDNA microarray data used in Stuart et al (Science 302(5643): 249-55 (2003)), and locally available SAGE data (elegans.bcgsc.ca), while the orthologues are obtained from WormBase and from WABA comparisons of C. elegans genes to unannotated genomic sequence of other nematodes (until their orthologues with C. elegans are published). Potential regulatory elements will be generated by using existing motif discovery algorithms, in a pipeline previously developed for mammalian genomes (www.cisred.org), to search for over-represented motifs in the upstream regions of the genes in each set. Regulatory elements will be predicted for each C. elegans gene for which appropriate coexpressed and orthologous genes are available. Known regulatory elements in C. elegans will be used as a positive control, while the upstream regions of sets of genes that are not coregulated will be used as a negative control. Putative regulatory elements will be validated in the lab via collaborations. Results from validations will be used to improve the next round of predictions in an iterative process.

Huang LS et al. (1990) Worm Breeder's Gazette "Molecular cloning of lin-15 by the candidate gene approach"

Sternberg PW, Huang LS, Tzou P, Mendel JE

[Worm Breeder's Gazette, 1990]

We have rescued the lin-15 multivulva.(Muv; visible or AB) and synthetic (syn; class A and class B) Muv phenotypes with cosmid PS#74B3. This cosmid maps to the far left (formerly, far right) end of the clb-1 contig on the X chromosome and was originally isolated because it weakly hybridizes to the G-protein PCR clone 1, as reported in WBG11(2):32. As we had been trying to clone lin-15 (see WBG11(3) :60 for last update), we knew from the map data that lin-15 should lie either at the right edge of the clb-1 contig or in the gap off the end. Since PS#74B3 appeared to map closely to lin-15 on the physical map and because the putative G-protein was a candidate gene for a genetic locus presumably involved in signal transduction, we decided to microinject the cosmid to test whether it rescued lin-15. As we had hoped, PS#74B3 rescued lin-15, although the G-protein hybridizing region did not rescue. A 'reverse northern' analysis identified a 6.8 kb BamHI fragment which lay within the region defined as necessary for lin-15 function by injection. When the 6.8 kb fragment was used to screen Stuart Kim and Chris Martin's cDNA libraries, we identified two classes of non-cross hybridizing cDNAs which mapped to opposite sides of our 6.8 kb fragment, with cDNA2 apparently more abundant than cDNA1.

Rosoff ML et al. (1991) International C. elegans Meeting "MOLECULAR CHARACTERIZATION OF THE NEUROPEPTIDE GENE ENCODING FLRFamide."

Li C, Rosoff ML

[International C. elegans Meeting, 1991]

Single neurons can express multiple neurotransmitters and in addition, can switch their transmitter phenotype during development. We are interested in identifying what factors may be involved in determining transmitter specificity. Approximately thirty neurons in C. elegans stain with an antibody specific for the RFamide moiety of the neuropeptide FMRFamide [see WBG 9(3):110]. Screening of Stuart Kim's cDNA library with degenerate oligonucleotides against FMRFGK(R) yielded a 659 bp hybridizing clone whose putative translation product encodes eight potential neuropeptides. Seven of these neuropeptides share the same carboxy terminal -PNFLRFamide structure. Primer extension analysis has shown that this cDNA is not full length. Since screening of the MIT and MRC cosmid libraries yielded no hybridizing clones, we screened a lambda 1059 genomic library ( courtesy of Scott Emmons), and isolated thirteen hybridizing clones. By restriction mapping, the clones were separated into four overlapping classes. A hybridizing 3 kb EcoRI fragment was subcloned and sequenced from two separate isolates. The clones contain approximately 300bp upstream of the start of the cDNA, the entire cDNA, and a poly adenylation signal. Constructs are being made to analyze the promoter region using the beta-galactosidase gene as a reporter in microinjection studies. Concurrently, we have begun the purification of FMRFamide-like peptides from worm extracts. Radioimmunoassays show the presence of FMRFamidelike peptides, and further characterization of the extracts by reverse phase HPLC reveals at least five peaks of FMRFamide-like immunoreactivity.

Kara Thoemke et al. (2001) International C. elegans Meeting "Identification and characterization of putative male-specific sexual regulators and TRA-1 target genes"

Woelsung Yi, Jennifer Ross, Kara Thoemke, David Zarkower, Marc Sohrmann

[International C. elegans Meeting, 2001]

C. elegans is highly sexually dimorphic, with about 30% of the somatic cells in the adult hermaphrodite and 40% of the somatic cells in the adult male being sexually specialized. In C. elegans , all aspects of somatic sexual development are controlled by TRA-1A, a zinc finger transcription factor. Sexual dimorphism in C. elegans affects all tissue types and is achieved through diverse mechanisms including sex-specific cell divisions and migrations, sex-specific cell death, generation of sex-specific blast cells and sex-specific changes in gene expression. TRA-1A probably directly regulates multiple genes involved in restricted aspects of sexual differentiation, and an important goal is to identify these genes. We have identified a large number of male-enriched genes in C. elegans using microarray analysis with help from Stuart Kim and the Stanford Microarray Database curators. We have compared N2 XX hermaphrodites and tra-2(ts) XX pseudomales from the L2 through the L4 stages to generate a developmental profile of sex-specific gene expression. Many of these genes likely function as male-specific sexual regulators. We have also identified putative TRA-1A binding sites in the C. elegans genome using a hidden Markov model (HMM) computer algorithm and weight matrix searches. Combining these two approaches, we seek to identify and characterize male-specific TRA-1A target genes in an effort to understand the mechanisms by which C. elegans achieves sexual dimorphism. We are currently conducting functional analysis of these putative male sexual regulators using RNAi and GFP reporter analysis.