[
Ann N Y Acad Sci,
1959]
A detailed summary of methods for axenic cultivation of Caenorhabditis briggsae is given. Results of axenic culture on chemically defined basal media (GM and GS) and on these media supplemented with undefined preparations of horse liver and chick embryos are reported in detail, with a review of the formulation of the GM and GS designs and of the chronology of changes made therein. The best growth so far realized with C. briggsae in axenic culture is suboptimal as compared with growth in the presence of bacteria, and maturation takes longer (4 to 5 days instead of about 3 days at 20C). Suitable media of the GM design give good axenic growth with relatively low levels of complex supplements-Liver Protein Fraction C (LPF-C) and chick embryo extract (CEE), both of which presumably include a protein-linked requirement, Factor Rb. With GM-16 plus CEE or certain GSs plus CEE, requirements have been variously demonstrated for 6 B-vitamins: folic acid, niacinamide, pantothenic acid, pyridoxine, riboflavin, and thiamine; one of these-folic acid-had already been shown to be required. Only niacinamide is also demonstrated as a requirement in the presence of low levels of LPF-C. In the presence of CEE we have tested the essentiality of the other 5 vitamins only by omitting them singly from vitamin mixes added at increased (5 to 50 times GS) levels to media of GM or GS type. Preliminary evidence is given that the ten "rat-essential" amino acids are required. Improvement of nutritional balance with respect to amino acid levels and to relative levels of amino acids in relation to vitamins or salts is discussed as an explanation of differential growth on different media. Possibly the variations of DM-GS so far tested contain unnecessarily high amino acid levels. The definition of nutritional requirements for C. briggsae still presents many
[
J Exp Zool,
1999]
A memorable workshop, focused on causal mechanisms in metazoan evolution and sponsored by NASA, was held in early June 1998, at MBL. The workshop was organized by Mike Levine and Eric H. Davidson, and it included the PI and associates from 12 different laboratories, a total of about 30 people. Each laboratory had about two and one half hours in which to represent its recent research and cast up its current ideas for an often intense discussion. In the following we have tried to enunciate some of the major themes that emerged, and to reflect on their implications. The opinions voiced are our own. We would like to tender apologies over those contributions we have not been able to include, but this is not, strictly speaking, a meeting review. Rather we have focused on those topics that bear more directly on evolutionary mechanisms, and have therefore slighted some presentations (including some of our own), that were oriented mainly toward developmental processes. J. Exp. Zool. (Mol. Dev. Evol. ) 285:104-115, 1999.
[
International Review of Cytology,
1986]
The problem of cell-specific gene expression has long been a major concern to developmental biologists. Why and how specific genes are expressed only in certain differentiated cells and not in others are of vital importance. Many well-documented examples of differentiated cell types expressing quantitative and/or qualitative changes in gene expression now exist. For example, Galau et al. (1976) demonstrated that different sets of genes are expressed during development and in different adult tissues of the sea urchin. More recently, Angerer and Davidson (1984) have used in situ hybridization of specific DNA probes to demonstrate the expression of lineage-specific genes long before morphological differentiation. Other examples include the ovalbumin gene, known to be expressed only in hormone-stimulated oviducts, and the globin genes expressed at various developmental stages in differentiating erythrocytes. Many other examples of cell-specific gene expression are known, including the silk moth chorion proteins, the glue proteins in Drosophila, and a-amylase in mammals. Detailed molecular analysis of genes has provided important information on the mechanisms of gene expression. For example, numerous studies have examined the role of chromatin structure as well as the significance of specific sequences in the transcription and translation of eukaryotic genes. Furthermore, studies of the globin, actin, immunoglobulin, histone, and silk moth chorion genes have demonstrated the existence of gene families with suggested importance for the evolution of new functions for old genes. In addition, the detailed study of multigene families has provided vital information on the mechanisms of cell-specific gene expression as seen, for example, in the temporal and spatial regulation of different members of the actin gene family....
[
Sci STKE,
2000]
Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arf's effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arf's role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events.
[
WormBook,
2006]
Heterotrimeric G proteins, composed of alpha , beta , and gamma subunits, are able to transduce signals from membrane receptors to a wide variety of intracellular effectors. In this role, G proteins effectively function as dimers since the signal is communicated either by the G alpha subunit or the stable G betagamma complex. When inactive, G alpha -GDP associates with G betagamma and the cytoplasmic portion of the receptor. Ligand activation of the receptor stimulates an exchange of GTP for GDP resulting in the active signaling molecules G alpha -GTP and free G betagamma , either of which can interact with effectors. Hydrolysis of GTP restores G alpha -GDP, which then reassociates with G betagamma and receptor to terminate signaling. The rate of G protein activation can be enhanced by the guanine-nucleotide exchange factor, RIC-8 , while the rate of GTP hydrolysis can be enhanced by RGS proteins such as EGL-10 and EAT-16 . Evidence for a receptor-independent G-protein-signaling pathway has been demonstrated in C. elegans early embryogenesis. In this pathway, the G alpha subunits GOA-1 and GPA-16 are apparently activated by the non-transmembrane proteins GPR-1 , GPR-2 , and RIC-8 , and negatively regulated by RGS-7 . The C. elegans genome encodes 21 G alpha , 2 G beta and 2 G gamma subunits. The alpha subunits include one ortholog of each mammalian G alpha family: GSA-1 (Gs), GOA-1 (Gi/o), EGL-30 (Gq) and GPA-12 (G12). The remaining C. elegans alpha subunits ( GPA-1 , GPA-2 , GPA-3 , GPA-4 , GPA-5 , GPA-6 , GPA-7 , GPA-8 , GPA-9 , GPA-10 , GPA-11 , GPA-13 , GPA-14 , GPA-15 , GPA-16 , GPA-17 and ODR-3 ) are most similar to the Gi/o family, but do not share sufficient homology to allow classification. The conserved G alpha subunits, with the exception of GPA-12 , are expressed broadly while 14 of the new G alpha genes are expressed in subsets of chemosensory neurons. Consistent with their expression patterns, the conserved C. elegans alpha subunits, GSA-1 , GOA-1 and EGL-30 are involved in diverse and fundamental aspects of development and behavior. GOA-1 acts redundantly with GPA-16 in positioning of the mitotic spindle in early embryos. EGL-30 and GSA-1 are required for viability starting from the first larval stage. In addition to their roles in development and behaviors such as egg laying and locomotion, the EGL-30 , GSA-1 and GOA-1 pathways interact in a network to regulate acetylcholine release by the ventral cord motor neurons. EGL-30 provides the core signals for vesicle release, GOA-1 negatively regulates the EGL-30 pathway, and GSA-1 modulates this pathway, perhaps by providing positional cues. Constitutively activated GPA-12 affects pharyngeal pumping. The G alpha subunits unique to C. elegans are primarily involved in chemosensation. The G beta subunit, GPB-1 , as well as the G gamma subunit, GPC-2 , appear to function along with the alpha subunits in the classic G protein heterotrimer. The remaining G beta subunit, GPB-2 , is thought to regulate the function of certain RGS proteins, while the remaining G gamma subunit, GPC-1 , has a restricted role in chemosensation. The functional difference for most G protein pathways in C. elegans, therefore, resides in the alpha subunit. Many cells in C. elegans express multiple G alpha subunits, and multiple G protein pathways are known to function in specific cell types. For example, Go, Gq and Gs-mediated signaling occurs in the ventral cord motor neurons. Similarly, certain amphid neurons use multiple G protein pathways to both positively and negatively regulate chemosensation. C. elegans thus provides a powerful model for the study of interactions between and regulation of G protein signaling.