Schneider SQ et al. (2003) Annu Rev Genet "Cell polarity and the cytoskeleton in the Caenorhabditis elegans zygote."

Bowerman B, Schneider SQ

[Annu Rev Genet, 2003]

The anterior-posterior axis of the Caenorhabditis elegans zygote forms shortly after fertilization when the sperm pronucleus and its associated centrosomal asters provide a cue that establishes the anterior-posterior (AP) body axis. In response to this cue, the microfilament cytoskeleton polarizes the distribution of a group of widely conserved, cortically localized regulators called the PAR proteins, which are required for the first mitotic division to be asymmetric. These asymmetries include a posterior displacement of the first mitotic spindle and the differential segregation of cell-fate determinants to the anterior and posterior daughters produced by the first cleavage of the zygote. Here we review recent advances in our understanding of the mechanisms that polarize the one-cell zygote to generate an AP axis of asymmetry.

Schneider SQ et al. (2003) J Exp Zool B Mol Dev Evol "Protein evolution: structure-function relationships of the oncogene beta-catenin in the evolution of multicellular animals."

Martindale MQ, Finnerty JR, Schneider SQ

[J Exp Zool B Mol Dev Evol, 2003]

Beta-catenin functions as a cytoskeletal linker protein in cadherin-mediated adhesion and as a signal mediator in wnt-signal transduction pathways. We use a novel integrative approach, combining evolutionary, genomic, and three-dimensional structural data to analyze and trace the structural and functional evolution of beta-catenin genes. This approach also enabled us to examine the effects of gene duplication on the structure and function of beta-catenin genes in Drosophila, C. elegans, and vertebrates. By sampling a large number of different taxa, we identified both ancestral and derived motifs and residues within the different regions of the beta-catenin proteins. Projecting amino acid substitutions onto the three- dimensional structure established for mouse beta-catenin, we identified specific domains that exhibit loss and gain of selective constraints during beta catenin evolution. Structural changes, changes in the amino acid substitution rate, and the appearance of novel functional domains in beta-catenin can be mapped to specific branches on the metazoan tree. Together, our analyses suggest that a single, beta-catenin gene fulfilled both adhesion and signaling functions in the last common ancestor of metazoans some 700 million years ago. In addition, gene duplications facilitated the evolution of beta-catenins with novel functions and allowed the evolution of multiple, single-function proteins (cell adhesion or wnt-signaling) from the ancestral, dual-function protein. Integrative methods such as those we have applied here, utilizing the ''natural experiments'' present in animal diversity, can be employed to identify novel and shared functional motifs and residues in virtually any protein among the proteomes of model systems and humans.

Yasuda H et al. (1988) Worm Breeder's Gazette "a tubulin tale from Toyohashi: two genes one polypedtide."

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1988]

Previously we have described our efforts to clone tubulin genes by direct screening of C. elegans expression libraries using nerve specific anti-tubulin antibodies(WBG 10, 1:59, 1987). For alpha- tubulin genes, 14 lambda gt11 clones were identified with a monoclonal antibody 3A5 (kindly given by M. Fuller), which is specific for alpha- tubulin and stains C. elegans neurons. Two of the 14 clones were subcloned in pUC 118, and sequenced by dideoxy method of Sanger and Coulson. One of these clones SQ#TbA77 (insert size 1.0 Kb) contained a DNA which is 79% homologous to the chicken alpha-tubulin DNA sequence; and its amino acid sequence deduced from DNA sequence is 88% homologous to the C-terminal region of the chicken alpha-tubulin. The second putative clone on sequencing revealed DNA that has no homology with tubulin DNA. Similarly, we have identified 15 lambda gt11 clones using E6B6, ( kind gift of T. Arai) a monoclonal antibody which is specific for alpha-tubulin and also stains C. elegans nervous system. Three of the 15 clones were subcloned and sequenced as stated above. One of these SQ#TbB03 has a sequence 76% homologous to the chicken tubulin DNA. We compared the nucleotide sequence of SQ#TbB03 with Nw#B8, the only other -tubulin cloned and sequenced in C. elegans (L. Gremke, Ph.D. Thesis,1987). The two nucleotide sequences and the deduced amino acid sequence is given below. It is quite remarkable that the two -tubulin genes have 100% homology at the level of amino acid sequence. Currently, we are trying to get the complete DNA sequence data of the tubulins identified by SQ#TbA77 and SQ#TbB03. [See Figure 1]

Fields CA (1990) Nucleic Acids Res "Information content of Caenorhabditis elegans splice site sequences varies with intron length."

Fields CA

[Nucleic Acids Res, 1990]

A database of sequences of 139 introns from the nematode Caenorhabditis elegans was analyzed using the information measure of Schneider et al. (1986) J. Mol. Biol. 128: 415-431. Statistically significant information is encoded by at least the first 30 nt and last 20 nt of C. elegans introns. Both the quantity and the distribution of information in the 5' splice site sequences differs between the typical short (length less than 75 nt) and rarer long (length greater than 75 nt) introns, with the 5 sites of long introns containing approximately one bit more information. 3' splice site sequences of long and short C. elegans introns differ significantly in the region between -20 and -10 nt.

Robin Schneider et al. (2006) European Worm Meeting "The Loss of the Single APC Homolog apr-1 Causes Hyperplasia in the C. elegans Intestine"

Olaf Bossinger, Gisela Helbig, Robin Schneider

[European Worm Meeting, 2006]

Robin Schneider, Gisela Helbig and Olaf Bossinger. In humans APC is a powerful tumour suppressor that is mutated in most cases of sporadic or congenital colorectal cancer. In these forms of cancer, cells lose their ability to control the cell cycle and cell migration along the crypt-villus axis is disturbed. The C. elegans intestine is a simple epithelial tube that consists of only 20 cells (E-cells). Depletion of the C. elegans APC homolog APR-1 (Hoier et al., 2000) by bacterial RNAi causes hyperplasia of E-cells. In contrast, apr-1(RNAi) embryos contain up to 40 E-cells, which in most cases achieve to arrange into a tube-like structure. The use of gut-specific RNAi against APR-1 and the gain-of-function phenotype of the cycline dependent kinase CDC-25.1 (Clucas et al., 2002) suggests that intestinal cell proliferation is under organ-specific control. Preliminary data indicate that C. elegans homologs of the vertebrate WNT pathway are also involved in the regulation of endodermal cell proliferation. A detailed analysis of gut-specific hyperplasia phenotypes will be presented and discussed with regard to the involved pathways.

Yasuda H et al. (1990) Worm Breeder's Gazette "Trans-splicing in Tubulin?"

Siddiqui SS, Yasuda H

[Worm Breeder's Gazette, 1990]

At the last worm meeting (CSH, 1989), we reported the nucleotide sequence of the alpha-1 tubulin gene AT3 that was cloned by direct screening of a C. elegans cDNA expression library in a lambda gt11 vector (Bob Barstead), using a tubulin specific monoclonal anti-body 3A5 (provided by M. Fuller). This gene was previously identified by Linda Gremke using a chicken tubulin probe (Don Cleveland). Our clone SQ#TbA77 was cytologically mapped on chromosome I of C. elegans (Alan Coulson and John Sulston, MRC, LMB, UK). As Dr. Tom Blumenthal walked by our poster at the meeting, he pointed out the presence of an eight nucleotide non-coding sequence TTCAGGTT, which corresponds to the acceptor sequence of C. elegans in the trans-splicing reaction of mRNA. This is a rather novel feature for the tubulin gene, as the other alpha or beta tubulin genes, from C. elegans or other metazoans have not been reported to be trans-spliced at the mRNA level. We are currently trying to sequence the mRNA to test if this mRNA is indeed spliced. Figure below shows the physical map of the alpha-1 tubulin gene, and the nucleotide sequence showing the acceptor sequence ( marked by arrowed box), in the non-coding sequence of the tubulin gene. [See Figure 1]

Rendi D et al. (2007) Electrophoresis "Adaptation of the "in-gel release method" to N-glycome analysis of low-milligram amounts of material."

Leonard R, Lubec G, Altmann F, Rendi D, Gutternigg M, Wilson IB

[Electrophoresis, 2007]

Protein N-glycosylation is a post-translational modification which plays numerous crucial physiological roles. The N-glycan pattern varies depending on the species organs, tissues and even cell types and their respective physiological states. Obtaining enough starting material from a particular cell type or tissue for N-glycan purification by conventional methods can, in certain cases, be very difficult. Previously, a sensitive technique, the "in-gel release method" that allows the determination of N-glycans attached to a protein isolated by SDS-PAGE, has been developed in this and other laboratories. Here, we describe the adaptation of this method to obtain information on the N-glycome from minute amounts of tissue. The starting material, ranging from less than a milligram to a few milligrams of fresh tissue, is directly ground in Laemmli sample buffer and subject briefly to discontinuous Tris-glycine-SDS-PAGE. The Coomassie-stained band containing the majority of the proteins is subject to the "in-gel release method". The developed technique was used to analyze N-glycan patterns of different samples from Caenorhabditis elegans, Drosophila melanogaster, Spodoptera frugiperda, Trichoplusia ni, Nicotiana benthamiana, Arabidopsis thaliana, and Mus musculus. Furthermore, the technique was used to determine the effects of transient small-scale RNAi-mediated knock-down of a glycosylation-related gene in Drosophila Schneider 2 cell line.

Li W et al. (2015) J Biol Chem "Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1."

Koutmou KS, Li W, Li M, Leahy DJ

[J Biol Chem, 2015]

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.

Christopher W Brey et al. (2001) International C. elegans Meeting "ENHANCEMENT OF STEINERNEMA CARPOCAPSAE DESICCATION TOLERANCE BY GENETIC IMPROVEMENT"

Randy Gaugler, Christopher W Brey

[International C. elegans Meeting, 2001]

The entomopathogenic nematode (ENP) Steinernema carpocapsae , is an important biological control agent for a wide range of soil dwelling insect pests. However, the field efficacy of this ENP is limited by its sensitivity to high drought and salinity conditions. We report efforts to improve the desiccation tolerance of S. carpocapsae by transforming it with the trehalose-6-phosaphate synthase (tps1) and glycogen synthase(gsy1)genes. Trehalose-6-phosphate synthase and glycogen synthase are enzymes involved in the biosynthesis of trehalose, a disaccharide that accumulates to stabilize the lipid biomembranes in many organisms when in response to stress. To increase desiccation tolerance by genetic modification, we have cloned gene tps1 from yeast and C. elegans into expression vectors pJJ436 and pPD95.67, respectively. In addition, we also cloned gsy1 from Steinernema feltiae into expression vector pJJ436. Vector pJJ436 contained the Ce sq-1 promoter, whereas pPD95.67 contained the promoter of the tps1 Ce gene. All vectors contained the gfp transformation gene which was used as a selection marker. Vector constructions (yeast: pJYeTr.1; C. elegans : pP67CeTr.2; S. feltiae : pJSfTr.1) were microinjected independently into young S. carpocapsae females (48 h from infective juvenile stage). Injected females were mated with noninjected males for 2-4 days and progeny were screened for gfp expression. After selecting and retaining gfp expressing individuals for three generations, F3 progeny were tested for desiccation tolerance. We will present details of our methodology and results in our presentation.

Nathalie Velarde et al. (2005) International Worm Meeting "F-actin dynamics in early C. elegans development"

Nathalie Velarde, Fabio Piano

[International Worm Meeting, 2005]

Actin has been shown to play key roles during early development in the C. elegans embryo (Schneider and Bowerman, 2003). However, it has been difficult to faithfully reproduce F-actin dynamics in vivo during early embryogenesis. To visualize F-actin we have generated a transgenic line that uses the F-actin binding domain of Drosophila moesin to decorate endogenous actin filaments with GFP (GFP::Moe). The GFP::Moe fusion line appears to be specific for filamentous actin and allows the visualization of F-actin dynamics in C. elegans in embryos. Preliminary evidence shows that F-actin is very dynamic in many cellular processes from prior to fertilization through the first mitotic cellular division. During fertilization a posterior actin cap forms and then dissipates. Prior to the first cell division F-actin becomes enriched in the anterior, similar to the anterior PAR proteins. As seen in par-3 mutants (Kirby et. al., 1990), depleting the embryo of PAR-6 with RNAi prevents this enrichment, suggesting that PAR-6 and the other anterior PARs may be required for F-actin to accumulate in the anterior. We have also observed the presence of highly dynamic actin comets in the early embryo. Preliminary evidence suggests that these comets may be involved with endocytic processes. Using results from large-scale RNAi surveys, we are employing the GFP::Moe line to perform secondary screening of genes associated with pseudocleavage defects to further characterize this process. We will present our progress in using this GFP::Moe line to study genetic interactions involving actin dynamics in early development.