Two components of the germ-line-specific P granules of the nematode Caenorhabditis elegans have been identified using polyclonal antibodies specific for each. Both components are putative germ-line RNA helicases (GLHs) that contain CCHC zinc fingers of the type found in the RNA-binding nucleocapsid proteins of retroviruses. The predicted GLH-1 protein has four CCHC fingers; GLH-2 has six. Both GLH proteins localize in the P granules at all stages of germ-line development. However, the two glh genes display different patterns of RNA and protein accumulation in the germ lines of hermaphrodites and males. Injection of antisense glh-1 or glh-2 RNA into wild-type worms causes some offspring to develop into sterile adults, suggesting that either or both genes are required for normal germ-line development. As these very similar glh genes physically map within several hundred kilobases of one another, it seems likely that they represent a fairly recent gene duplication event.Embryos of the free-living soil nematode Caenorhabditis elegans generate distinct founder cells via a series of asymmetric cell divisions. At each division, the germ-line daughter cell inherits distinctive non-membrane-bound particles, called P granules (1-3). P granules are partitioned to the primordial germ cell P 4 of the 16-to 24-cell embryo and become perinuclear. P granules persist around the nuclei of all germ cells, until gametogenesis, at which point they are excluded from mature sperm and become dispersed within the cytoplasm of mature oocytes in preparation for cytoplasmic localization in the embryo. Although the distribution pattern of nematode P granules has been well-studied, the identity and function of P-granule components have yet to be determined.Germ granules are found in many species (4, 5). The germ-line-specific polar granules of Drosophila melanogaster have been well-studied, with a number of different genes identified that are required for polar granule assembly and germ-cell formation, including vasa, staufen, valois, oskar, tudor, mago nashi, and germ-cell-less (6-15). With the exception of vasa, these genes encode novel proteins. Vasa, however, is a member of a family of proteins with recognizable motifs and predictable function. Vasa is an RNA helicase of the DEAD-box family (8, 9) whose ATP-dependent RNA helicase activity has been demonstrated in vitro (16). As polar granules contain RNA as well as protein (11,12,15,17), a germ-linespecific RNA helicase may function to bind and unwind RNAs necessary for germ-line development. Several potential vasa homologues have been cloned, including glh-1 (germ-line helicase 1) from Caenorhabditis, Xvh (Xenopus vasa homologue), mvh (mouse vasa homologue), and rvh (rat vasa homologue) (18)(19)(20)(21). glh-1 in C. elegans is unique among RNA helicase genes reported, including vasa, in that its predicted product contains four retroviral-like zinc fingers (18). We have identified a second C. elegans germ-line RNA helicase gene, glh-2, that also encodes zinc fingers. Immunolocali...

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germcell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.

The forkhead transcription factor, DAF-16, a downstream target of the insulin/IGF-I signaling pathway in C. elegans, is indispensable both for lifespan regulation and stress resistance. The molecular mechanisms involved in regulating DAF-16 transcriptional activation remain undefined. Here, we have identified an E3 ubiquitin ligase, RLE-1 (regulation of longevity by E3), which regulates aging in C. elegans. Disruption of RLE-1 expression in C. elegans increases lifespan; this extension of lifespan is due to elevated DAF-16 protein but not to changes of daf-16 mRNA levels. We have also found that RLE-1 catalyzes DAF-16 ubiquitination, leading to degradation by the proteasome. Elimination of RLE-1 expression in C. elegans causes increased transcriptional activation and sustained nuclear localization of DAF-16. Overexpression of DAF-16 in rle-1 mutants increases worm lifespan, while disruption of DAF-16 expression in rle-1 mutants reverses their longevity. Thus, RLE-1 is an E3 ubiquitin ligase of DAF-16 that regulates C. elegans aging.

The addition of cosolvents to supercritical fluid (SCF) solvents can have large effects on solubilities, giving engineers the ability to tailor loadings and selectivities of solutes for difficult separations. It is necessary to have a better understanding of the special intermolecular interactions that occur in SCF solutions to predict the effects of cosolvents. We use u SCF chromatographic technique to acquire a database of cosolvent effects for a variety of cosolvents and solutes; examination of the cosolvent effects shows evidence of hydrogen bonding, charge transfer complex formation, and dipole-dipole coupling between solute and cosolvent molecules. SCF solvents, carbon dioxide, ethane, and fluoroform, are compared, and then the use of the chromatograph to measure solubilities is discussed.