Stuart K Kim et al. (2001) International C. elegans Meeting "A gene expression map for C. elegans"

Stewart Scherer, George S Davidson, Stuart K Kim, Jim Lund, Brian N Wylie, Min Jiang, Kyle Duke, Moni Kiraly

[International C. elegans Meeting, 2001]

The completion of the C. elegans genome sequence has identified nearly all of the genes in the genome (19,282 genes), but the function for most of these genes remains mysterious. A scant 6% of them have been studied using classical genetic or biochemical approaches (1135 genes), and only about 53% show homology to genes in other organisms (10,303 genes). The next challenge is to develop high-throughput, functional genomics procedures to study many genes in parallel in order to elucidate gene function on a global scale. One such approach is to use DNA microarrays to assay the relative expression of nearly every gene in the genome between two samples. Knowing when, where and under which conditions a particular gene is expressed can reveal the function of that gene. A consortium of laboratories has used C. elegans DNA microarrays to profile gene expression changes in a wide variety of experiments. In each experiment, RNA from one sample was used to generate Cy3-labelled cDNA, and RNA from another sample was used to prepare Cy5-labelled cDNA. The two cDNA probes were simultaneously hybridized to a single DNA microarray and the ratio of the Cy3 to Cy5 hybridization intensities was measured, revealing which genes were relatively enriched in either RNA sample. Thirty different laboratories have collectively performed 553 experiments using these C. elegans DNA microarrays, including 179 experiments with microarrays containing 11,917 genes (63% of the genome) and 370 experiments using microarrays that have 17,817 genes (94% of the genome). The experiments compare RNA between mutant and wild-type strains, or between worms grown under different conditions. Many experiments have been done to date, including experiments on wild-type development, heat shock, Ras signaling, aging, the dauer stage, sex regulation and germ line gene expression. Individual microarray experiments reveal sets of genes that change in one mutant strain or growth condition. We combine the data from all of the experiments to assemble a gene expression database, and then used this database to group together co-regulated genes and visualize them using a three dimensional expression map. By matching the expression profile of an unknown gene to those of genes with known functions, the expression map can be used to ascribe functions to the large fraction of genes in the genome whose functions were previously unknown.

Stuart K Kim et al. (1999) International C. elegans Meeting "Using DNA microarrays to identify targets of the Ras/MAP kinase signaling pathway"

Stuart K Kim, Carrie B Van Doren, Rebecca R Begley

[International C. elegans Meeting, 1999]

We are entering a new age in molecular genetics in which we can use sequence data from genome projects to dissect cell, developmental and disease pathways more completely and more sensitively than ever before. We need to develop new experimental approaches to analyze the vast amounts of data that are now available from genome projects. C. elegans is the only animal model system with a complete genome sequence, and thus will play a leading role in establishing approaches that make use of the full genome sequence. One key functional genomics approach is to use DNA microarrays to define changes in gene expression patterns during development and during the onset of disease. DNA microarrays could be used to identify genes that are regulated by specific transcription factors, by specific cell signaling pathways, by expression of homologs of human disease genes in transgenic animals or by addition of various pharmaceutical drugs. We are currently producing DNA microarrays that contain 11,401 genes from the C. elegans genome. To demonstrate that C. elegans microarrays are powerful tools to dissect developmental pathways, we are using these DNA microarrays to identify targets of the RTK/Ras/MAP kinase signaling pathway during vulval development. To globally detect differences in transcription patterns due to Ras signaling, a cDNA microarray is simultaneously hybridized with Cy3-labelled cDNA (red) (from reverse transcription of polyA mRNA) from let-23(lf) mutants, and Cy5-labelled cDNA (green) from let-60(gf) mutants. Genes that are expressed at similar levels in the two strains hybridize equally with both probes, and appear yellow on the microarray. Genes that are expressed at higher levels in the first strain appear red, and genes that are expressed at higher levels in the second strain appear green. The image intensities of the cDNA microarray are digitized, and the expression ratios between the two strains are calculated for every cDNA clone. The Ras target genes identified by the DNA microarrays will play a critical role in elucidating the molecular basis for Ras signaling. Once we have developed full genome C. elegans microarrays, we can make our DNA microarrays available to the entire C. elegans academic community.

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.

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.

Kendall Wu et al. (2004) West Coast Worm Meeting "Identification of Genetic Biomarkers and Regulators of Aging in C. elegans"

James Lund, Min Jiang, John Wang, Yueyi Liu, Stuart Kim, Kendall Wu

[West Coast Worm Meeting, 2004]

Despite the wealth of knowledge about conditions and mutations that cause worms to live longer, we still have an incomplete understanding of what varies from young to old worms -- especially at the molecular level. A long-term goal of our laboratory is to determine the molecular changes that occur as an organism ages in order to better understand the aging process and its regulation. Using the nematode Caenorhabditis elegans ( C. elegans ) as a genetic model for aging, we have used DNA microarrays to identify a common set of genes regulated throughout several age-related conditions: genes that change in old age (Lund et al. , 2002), genes that change in the exit from the dauer state (Wang and Kim, 2003), and genes that change expression in long-lived mutants of the insulin-like pathway such as the age-1 and daf-16 mutants (unpublished data). Analysis of the upstream regions of these age-regulated genes showed that they are heavily enriched for a GATA DNA consensus sequence suggesting that a GATA transcription factor may be involved in regulating the expression of these genes. This GATA motif may represent a novel regulatory pathway of the aging process that might act either together or separately from the daf-2 /insulin-like pathway. Using GFP reporters of these genes, we have begun to determine how these genes are age-regulated and to identify the key tissues regulating lifespan. We have also used RNAi to abrogate the expression of these genes to determine their role in longevity. The results of these studies will define a set of molecular biomarkers that will allow a more accurate description of the aging process. In addition, these biomarkers will allow identification of new aging mutants perhaps leading to identification of mutants with accelerated aging. Lund, J., Tedesco, P., Duke, K., Wang, J., Kim, S. K., and Johnson, T. E. (2002). Transcriptional profile of aging in C. elegans. Curr Biol 12, 1566-1573. Wang, J., and Kim, S. K. (2003). Global analysis of dauer gene expression in Caenorhabditis elegans. Development 130, 1621-1634.