Epigallocatechin gallate from green tea (Camellia sinensis) increases lifespan and stress resistance in Caenorhabditis elegans.

Epigallocatechin gallate (EGCG) is a major green tea polyphenol with pronounced antioxidative activity. The effects of EGCG on lifespan and stress resistance in wild-type N2 and transgenic strains of Caenorhabditis elegans [ HSP-16.2/GFP, MEV-1(KN1), FEM-1(HC17)] were investigated. The expression of HSP-16.2 (induced by the pro-oxidant juglone) and the intracellular levels of H (2)O (2) were inhibited by EGCG treatment. Daily administration of 220 muM EGCG increased the mean lifespan by 10.14 % and 14.27 % in N2 and FEM-1(HC17) strains, respectively, and 55 muM EGCG increased the mean lifespan in MEV-1(KN1) by 16.11 %. The survival rate was also increased under lethal oxidative stress by 65.05 %. These findings suggest that the increased mean lifespan and stress resistance in C. ELEGANS apparently depend, among other factors, on the antioxidant properties of EGCG.

XOL-1, primary determinant of sexual fate in C. elegans, is a GHMP kinase family member and a structural prototype for a class of developmental regulators.

In Caenorhabditis elegans, an X chromosome-counting mechanism specifies sexual fate. Specific genes termed X-signal elements, which are present on the X chromosome, act in a concerted dose-dependent fashion to regulate levels of the developmental switch gene xol-1. In turn, xol-1 levels determine sexual fate and the activation state of the dosage compensation mechanism. The crystal structure of the XOL-1 protein at 1.55 A resolution unexpectedly reveals that xol-1 encodes a GHMP kinase family member, despite sequence identity of 10% or less. Because GHMP kinases, thus far, have only been characterized as small molecule kinases involved in metabolic pathways, for example, amino acid and cholesterol synthesis, XOL-1 is the first member that controls nonmetabolic processes. Biochemical investigations demonstrated that XOL-1 does not bind ATP under standard conditions, suggesting that XOL-1 acts by a mechanism distinct from that of other GHMP kinases. In addition, we have cloned a XOL-1 ortholog from Caenorhabditis briggsae, a related nematode that diverged from C. elegans approximately 50-100 million years ago. These findings demonstrate an unanticipated role for GHMP kinase family members as mediators of sexual differentiation and dosage compensation and, possibly, other aspects of differentiation and development.

Secretion of a novel class of iFABPs in nematodes: coordinate use of the Ascaris/Caenorhabditis model systems.

A novel fatty acid binding protein, As-p18, is secreted into both the perivitelline and perienteric fluids of the parasitic nematode, Ascaris suum, and at least eight potential homologues of As-p18 have been identified in the Caenorhabditis elegans genome. The products of the three most closely related homologues are fatty acid binding proteins (LBP-1, LBP-2 and LBP-3) which contain putative secretory signals. Phylogenetic analysis revealed that these secreted fatty acid binding proteins comprise a distinct gene class within the fatty acid binding protein family and are possibly unique to nematodes. To examine the potential sites of As-p18 secretion, the expression of the putative promoters of the C. elegans homologues was examined with GFP reporter constructs. The developmental expression of lbp-1 was identical to that of As-p18 and consistent with the secretion of LBP-1 from the hypodermis to the perivitelline fluid. The expression patterns of lbp-2 and lbp-3 were consistent with the secretion of LBP-2 and LBP-3 from muscle into the perienteric fluid later in development. These studies demonstrate that at least some perivitelline fluid proteins appear to be secreted from the hypodermis prior to the formation of the cuticle and, perhaps more importantly, that this coordinate C. elegans/A. suum approach may be potentially useful for examining a number of key physiological processes in parasitic nematodes.

The cuticle of the nematode Caenorhabditis elegans: a complex collagen structure.

The cuticle of the nematode Caenorhabditis elegans forms the barrier between the animal and its environment. In addition to being a protective layer, it is an exoskeleton which is important in maintaining and defining the normal shape of the nematode. The cuticle is an extracellular matrix consisting predominantly of small collagen-like proteins that are extensively crosslinked. Although it also contains other protein and non-protein compounds that undoubtedly play a significant part in its function, the specific role of collagen in cuticle structure and morphology is considered here. The C. elegans genome contains between 50 and 150 collagen genes, most of which are believed to encode cuticular collagens. Mutations that result in cuticular defects and grossly altered body form have been identified in more than 40 genes. Six of these genes are now known to encode cuticular collagens, a finding that confirms the importance of this group of structural proteins to the formation of the cuticle and the role of the cuticle as an exoskeleton in shaping the worm. It is likely that many more of the genes identified by mutations giving altered body form, will be collagen genes. Mutations in the cuticular collagen genes provide a powerful tool for investigating the mechanisms by which this group of proteins interact to form the nematode cuticle.

The expression of two P-glycoprotein (pgp) genes in transgenic Caenorhabditis elegans is confined to intestinal cells.

P-glycoproteins can cause multidrug resistance in mammalian tumor cells by active extrusion of cytotoxic drugs. The natural function of these evolutionarily conserved, membrane-bound ATP binding transport proteins is unknown. In mammals, P-glycoproteins are abundantly present in organs associated with the digestive tract. We have studied the tissue-specific expression of Caenorhabditis elegans P-glycoprotein genes pgp-1 and pgp-3 by transformation of nematodes with pgp-lacZ gene fusion constructs in which the promoter area of the pgp genes was fused to the coding region of lacZ. Expression of pgp-1 and pgp-3, as inferred from pgp-lacZ transgenic nematodes, was confined to the intestinal cells. The expression patterns of both genes were virtually indistinguishable. Quantitative analysis of pgp mRNA levels during development showed that pgp-1, -2, and -3 were expressed throughout the life cycle of C.elegans, albeit with some variation indicating developmental regulation. The expression of P-glycoprotein genes in intestinal cells is an evolutionarily conserved feature of these genes, consistent with the hypothesis that P-glycoproteins provide a mechanism of protection against environmental toxins.

The mec-7 beta-tubulin gene of Caenorhabditis elegans is expressed primarily in the touch receptor neurons.

Mutants of the mec-7 beta-tubulin gene of Caenorhabditis elegans lack the large diameter 15-protofilament microtubules normally found only in the set of six touch receptor neurons. Both a mec-7-lacZ reporter gene and affinity-purified anti-mec-7 antibodies were used to show that mec-7 is expressed primarily in the touch neurons. These data are consistent with a possible instructive role for the mec-7 tubulin in determining microtubule protofilament number. The antibodies and the mec-7-lacZ transgene were also used to examine mec-7 expression in mutants affecting the generation, differentiation or maintenance of the touch neurons. Decreased expression was observed in mutants of unc-86 and mec-3, genes that encode transcription factors essential for touch receptor neuron generation and differentiation, respectively.

Gut-specific and developmental expression of a Caenorhabditis elegans cysteine protease gene.

A Caenorhabditis elegans cysteine protease gene fragment, amplified by PCR using conserved eukaryotic protease gene sequences as primers, was used as a probe to isolate cDNA and genomic clones. The genomic clone, which had a coding sequence of 987 bp interrupted by 2 small introns, was physically mapped to the middle of linkage group V. The predicted amino acid sequence of the mature C. elegans cysteine protease was homologous to those of other eukaryotic cysteine proteases, particularly to that of the nematode parasite Haemonchus contortus (50%) and to the cathepsin B-like hemoglobinase of the trematode parasite Schistosoma mansoni (54%). The pro region of the C. elegans protease was homologous only to that of the H. contortus enzyme, implying a similar mechanism of protease activation. The C. elegans cysteine protease gene was temporally regulated: abundant 1.1-kb transcripts were detected in larvae and adults, but not in embryos. Transcription also was spatially regulated, occurring only in the intestine. Like the vitellogenin genes, which also are transcribed exclusively in the intestine, the 5' end of the C. elegans cysteine protease gene had at least one copy of each of 2 heptameric sequences which may be transcriptional regulatory elements governing gut-specific expression.

Inactivation of wild-type and rad mutant Caenorhabditis elegans by 8-methoxypsoralen and near ultraviolet radiation.

Survival of wild-type and four radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans was determined after near-UV irradiation in the presence of 8-methoxypsoralen (8-MOP). Three sets of inactivation profiles were generated for each strain by irradiating synchronous populations of either early embryos, late embryos or first-stage larvae (L1s). Late embryos were consistently the most sensitive. Curiously, none of the four rad mutants were even moderately hypersensitive. Split-dose experiments indicated that DNA-DNA crosslinks were primarily responsible for lethality. Crosslink induction and repair were determined using two different assays. In both cases, little if any repair was observed in wild-type. This lack of repair thus explains why the rad mutants were not hypersensitive to 8-MOP photoinactivation. Since early embryos undergo extensive cell cycling, their resistance to 8-MOP photoinactivation suggests that replication is highly refractory to both monoadducts and crosslinks, as has been demonstrated previously for UV radiation-induced photoproducts (Hartman et al., 1991, Mutat. Res., 255, pp. 163-173).

Signal transduction during C. elegans vulval induction.

Nematode proteins related to the human epidermal growth factor receptor and Ras proteins act in a common pathway to control cell fates in response to an inductive signal. Analysis of these gene products during C. elegans vulval induction allows detailed study of their function in the context of a developing organism.

Metabolism of plant sterols by nematodes.

Parasitic nematodes do not biosynthesize sterols de novo and therefore possess a nutritional requirement for sterol, which must be obtained from their hosts. Consequently, the metabolism of phytosterols by plant-parasitic nematodes is an important process with potential for selective exploitation. The sterol compositions of several species of plant-parasitic nematodes were determined by capillary gas chromatography-mass spectrometry and compared with the sterol compositions of their hosts. Saturation of the phytosterol nucleus was the major metabolic transformation performed by the root-knot nematodes Meloidogyne arenaria and M. incognita and the corn root lesion nematode, Pratylenchus agilis. In addition to saturation, the corn cyst nematode, Heterodera zeae, dealkylated its host sterols at C-24. Because free-living nematodes can be cultured in sterol-defined artificial medium, they have been successfully used as model organisms for investigation of sterol metabolism in plant-parasitic nematodes. Major pathways of phytosterol metabolism in Caenorhabditis elegans, Turbatrix aceti and Panagrellus redivivus included C-24 dealkylation and 4 alpha-methylation (a pathway unique to nematodes). C. elegans and T. aceti introduced double bonds at C-7, and T. aceti and P. redivivus saturated the sterol nucleus similarly to the plant-parasitic species examined. Several azasteroids and long-chain dimethylalkylamines inhibited growth and development of C. elegans and also the delta 24-sterol reductase enzyme system involved in the nematode C-24 dealkylation pathway.

Multiple functions of let-23, a Caenorhabditis elegans receptor tyrosine kinase gene required for vulval induction.

The let-23 gene, which encodes a putative tyrosine kinase of the epidermal growth factor (EGF) receptor subfamily, has multiple functions during Caenorhabditis elegans development. We show that let-23 function is required for vulval precursor cells (VPCs) to respond to the signal that induces vulval differentiation: a complete loss of let-23 function results in no induction. However, some let-23 mutations that genetically reduce but do not eliminate let-23 function result in VPCs apparently hypersensitive to inductive signal: as many as five of six VPCs can adopt vulval fates, in contrast to the three that normally do. These results suggest that the let-23 receptor tyrosine kinase controls two opposing pathways, one that stimulates vulval differentiation and another that negatively regulates vulval differentiation. Furthermore, analysis of 16 new let-23 mutations indicates that the let-23 kinase functions in at least five tissues. Since various let-23 mutant phenotypes can be obtained independently, the let-23 gene is likely to have tissue-specific functions.

Kinesin-related gene unc-104 is required for axonal transport of synaptic vesicles in C. elegans.

unc-104 encodes a novel kinesin paralog that may act as a microtubule-based motor in the nervous system. Neuronal cell lineages and axonogenesis are normal in unc-104 null mutants, but axons have few synaptic vesicles and make only a few small synapses. By contrast, neuron cell bodies have surfeits of similar vesicles tethered together within the cytoplasm. Based on behavioral and cellular phenotypes, we suggest that UNC-104 is a neuron-specific motor used for anterograde translocation of synaptic vesicles along axonal microtubules. Other membrane-bounded organelles are transported normally.

Caenorhabditis elegans ras gene let-60 acts as a switch in the pathway of vulval induction.

The let-60 gene, an essential ras gene of the nematode Caenorhabditis elegans, acts as a switch in the inductive signalling pathway that initiates vulva formation. Recessive let-60 mutations that cause a vulvaless phenotype prevent let-60 function in response to the inductive signal. These mutations are clustered and define regions necessary either for the activation or for the action of the let-60 ras protein. Dominant let-60 mutations that cause a multivulva phenotype alter codon 13 and activate let-60 in vivo, rendering it independent of the inductive signal. The let-60 gene acts within an extensively defined genetic pathway, and other genes within this pathway seem likely to encode molecules that regulate let-60 function as well as molecules that are targets of let-60 action.

The let-23 gene necessary for Caenorhabditis elegans vulval induction encodes a tyrosine kinase of the EGF receptor subfamily.

The let-23 gene is required for induction of the Caenorhabditis elegans vulva. It is shown that let-23 encodes a putative tyrosine kinase of the epidermal growth factor receptor subfamily. Thus, let-23 might encode the receptor for the inductive signal required for vulval development. Because let-23 acts upstream of let-60 ras in the vulval determination pathway, the identification of the let-23 product provides support for a link in vivo between tyrosine kinase growth factor receptors and ras proteins in a pathway of cell-type determination.

daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase.

The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.

The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.

The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.

Casein kinase II from Caenorhabditis elegans. Properties and developmental regulation of the enzyme; cloning and sequence analyses of cDNA and the gene for the catalytic subunit.

The nematode Caenorhabditis elegans provides a model system for investigating the structure, function, and regulation of casein kinase II. Cytosols from C. elegans embryos and gravid adults, which contain fertilized eggs and embryos, are enriched in casein kinase II activity; cytosols from newly hatched larva, four subsequent larval stages, and immature adults exhibit casein kinase II levels that are 3-10-fold lower than those observed in embryo cytosol. C. elegans casein kinase II contains alpha (Mr = 42,000) and beta (Mr = 29,000) subunits and has a Stokes radius of 50 nm. The enzyme utilizes ATP and GTP as substrates, is potently inhibited by heparin and undergoes autophosphorylation. Sequence analyses of cloned cDNAs corresponding to the 1.7-kilobase mRNA encoding the alpha (catalytic) subunit of casein kinase II indicate that the alpha polypeptide contains 359 amino acid residues. Variations in the abundance of casein kinase II alpha mRNA are coordinated with changes in enzyme activity during C. elegans development, indicating that alpha subunit expression is controlled at a pretranslational level. However, the magnitude of the developmentally controlled changes in phosphotransferase activity exceeded the corresponding increments in alpha subunit mRNA content. This suggests that translational and/or post-translational mechanisms also play an important role in the developmental regulation of C. elegans casein kinase II activity. The 2.9-kilobase casein kinase II alpha gene is divided into eight exons by intervening sequences ranging from 48 to 457 base pairs in length. The alpha gene promoter contains a TATA box, and a unique transcription start site has been identified. The intron/exon organization of the casein kinase II alpha gene differs markedly from the gene structure of the catalytic subunit of murine cAMP-dependent protein kinase (Chrivia, J. C., Uhler, M. D., and McKnight, G. S. (1988) J. Biol. Chem. 263, 5739-5744).

The regulation of DNA repair during development.

DNA repair is important in such phenomena as carcinogenesis and aging. While much is known about DNA repair in single-cell systems such as bacteria, yeast, and cultured mammalian cells, it is necessary to examine DNA repair in a developmental context in order to completely understand its processes in complex metazoa such as man. We present data to support the notion that proliferating cells from organ systems, tumors, and embryos have a greater DNA repair capacity than terminally differentiated, nonproliferating cells. Differential expression of repair genes and accessibility of chromatin to repair enzymes are considered as determinants in the developmental regulation of DNA repair.