David S Peal et al. (2003) International Worm Meeting "Negative Regulation of FGF Signaling By Heparan Sulfate in Caenorhabditis elegans"

David S Peal, Laura L Kiessling, Beth Stadtmueller, Judith Kimble

[International Worm Meeting, 2003]

Heparan sulfate (HS) proteoglycans are proteins that display sulfated carbohydrate polymers. HS proteoglycans are implicated in numerous biological processes, including signal transduction and modulation of proteolytic cascades. The 3-O sulfate group found on the N-acetylglucosamine moiety of HS is particularly intriguing. The only known role of this group is to modulate the coagulation cascade in mammals, yet multiple genes encoding 3-O sulfotransferases and their spliced isoforms are expressed in complex and tissue-specific patterns during development. These observations imply that 3-O sulfation may have other functions. To investigate the role of 3-O sulfation, we have isolated a null mutant of the 3-O sulfotransferase (3OST) gene in C. elegans. This mutant is clear (clr) at a low penetrance (35%). This previously described phenotype is caused by upregulation of the FGF signaling pathway(1). Our results suggest that the 3-O sulfate group of HS is involved in negative regulation of FGF signaling. We are testing this hypothesis through genetic and biochemical experiments. (1) Kokel, M, Borland, CZ, DeLong, L, Horvitz, HR, Stern, MJ. Genes Dev 1998; 12: 1425-37.

Chenggang Zhang et al. (2008) "Estimation of error rate of the genome-wide RNAi screening in Caenorhabditis elegans by analyzing non-specificity amplification of the PCR primers using the PSC program"

Wubin Qu, Zhiyong Shen, Chenggang Zhang, Bin Ning

[2008]

"Abstract: RNA interference (RNAi) is an efficiency and widely used technique in the functional genomics research. Genome-wide RNAi screening library for Caenorhabditis elegans is commercially available to advance large-scale analysis of the function for most (86%) of the 19,000 predicted genes. Although the RNAi screening library was constructed based on polymerase chain reaction (PCR) without directly sequencing to validate the nearly 16,000 gene sequences, however, the error introduced by PCR will probably occur during the preparation of the library and in turn will result in interfere of other unexpected genes. By using a recently developed computer program named Primer Specificity Checking (PSC, http://biocompute.bmi.ac.cn/PSC), the PCR primers for the 16,000 genes were analyzed carefully to determine whether they will result in mis-targeting the expected genes. Results indicated that at least 12.5% of the PCR primers have predominant non-specific amplifications. Most of the typical non-specific amplifications were caused by the primer-pairs capable to amplify different target genes in different chromosome location, while a minor proportion of the non-specific amplifications were caused by the self-amplification of only one primer. This will result in nearly 2,000 genes failed in the RNAi experiment. In conclusion, we suggested the user to check the primer sequences to avoid the error caused by PCR during the genome-wide RNAi screening in Caenorhabditis elegans. Keyword: Genome-wide RNAi screening; non-specificity amplification; Primer; PCR; PSC *Correspondence should be addressed to CZ (Email: zhangcg@bmi.ac.cn; Tel: +8610-66931590. Fax: +8610-68169574)."

Wu S-L et al. (1996) East Coast Worm Meeting "CHARACTERIZATION OF AN ATYPICAL PROTEIN KINASE C IN C. Elegans."

Rubin CS, Wu S-L

[East Coast Worm Meeting, 1996]

Protein kinase Cz (PKCz) is an atypical member of the PKC superfamily. PKCz contains Cys-rich zinc binding and catalytic domains that are homologous with corresponding regions in other PKCs. Unlike other PKCs, the zeta isoform in not activated by diacyl glycerol, TPA or calcium. However, PKCz appears to be a target for activation by alternative lipid second messengers (3,4,5-phosphoinositol, ceramide) generated by PI-3-kinase and/or sphingomyelinase. Activated PKCz has been implicated in the transmission of regulatory signals from the cytoplasm to the nucleus, but its precise physiological roles remain to be rigorously determined. We have initiated studies on the structure, function and regulation of an atypical C. elegans PKC (designated PKC-Z) that is related to mammalian PKCz. A cDNA that encodes full-length PKC-Z was cloned and sequenced. The predicted PKC-Z protein (598 amino acid residues, molecular weight = 68,000) is only ~55% identical with mammalian PKCz. N-terminal and central portions of the two proteins are highly divergent. However, the catalytic, psuedosubstrate and Cys-rich domains are highly conserved. Comparison of the cDNA sequence with data from the C. elegans genome project indicates that the PKC-Z gene (LGII) encompasses 3.6 kbp of DNA and contains 9 exons. A C terminal fragment (120 amino acids) of PKC-Z was expressed as a His-tagged fusion protein in E. coli, purified and injected into rabbits to produce antiserum. Western immunoblot analysis revealed that PKC-Z is most abundantly expressed in the soluble fraction of embryos. The kinase is also evident in L1-L4 larvae and adult nematodes, but the bulk of the enzyme is tightly associated with the particulate fractions of post-embryonic animals. Consistent with the latter observation, immunostaining and confocal microscopy of permeabilized C. elegans revealed that PKC-Z is localized and concentrated in discrete intracellular clusters. The nature of the intracellular structure at which PKC-Z accumulates is not yet known. PKC-Z is being expressed in baculovirus/Sf9 cells and in mammalian cells for enzymic characterization. In addition, injected anti-sense oligonucleotides are being used to probe the function(s) of PKC-Z in embryogenesis.