The spliceosomal snRNAs of Caenorhabditis elegans.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.     

Role of cell-mediated immunity in the resistance to experimental sporotrichosis in mice

The in vitro activity of several new imidazoles, cloconazole, sulconazole, butoconazole, isoconazole and fenticonazole, were compared with those of amphothericin B, flucytosine, and three azoles: econazole, miconazole and ketoconazole against isolates of pathogenic Candida. A total of 186 clinical isolates of 10 species of the genus Candida and two culture collection strains were tested by an agar-dilution technique. Isoconazole was the most active azole, followed by butoconazole and sulconazole. Differences between some of the species in their susceptibility to the antifungal agents were noted. Sulconazole and cloconazole had the highest activity in vitro against 106 isolates of C. albicans. Butoconazole and isoconazole were also very active against isolates of C. albicans, and were the most active azole compounds against 80 isolates of Candida spp.     

Regulated expression of a vitellogenin fusion gene in transgenic nematodes

In Caenorhabditis elegans the vitellogenin genes are expressed abundantly in the adult hermaphrodite intestine, but are otherwise silent. In order to begin to understand the mechanisms by which this developmental regulation occurs, we used the transformation procedure developed for C. elegans by A. Fire (EMBO. J., 1986, 5, 2673-2680) to obtain regulated expression of an introduced vitellogenin fusion gene. A plasmid with vit-2 upstream and coding sequences fused to coding and downstream sequences of vit-6 was injected into oocytes and stable transgenic strains were selected. We obtained seven independent strains, in which the plasmid DNA is integrated at a low copy number. All strains synthesize substantial amounts of a novel vitellogenin-like polypeptide of 155 kDa that accumulates in the intestine and pseudocoelom, but is not transported efficiently into oocytes. In two strains examined in detail the fusion gene is expressed with correct sex, tissue, and stage specificity. Thus we have demonstrated that the nematode transgenic system can give proper developmental expression of introduced genes and so can be used to identify DNA regulatory regions.     

pH-dependent lysis of liposomes by adenovirus

Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle.     

Characterization of the phosphotyrosyl protein phosphatase activity of calmodulin-dependent protein phosphatase

Calmodulin-dependent protein phosphatase from bovine brain and heart was assayed for phosphotyrosine and phosphoserine phosphatase activity using several substrates: 1) smooth muscle myosin light chain (LC20) phosphorylated on tyrosine or serine residues, 2) angiotensin I phosphorylated on tyrosine, and 3) synthetic phosphotyrosine- or phosphoserine-containing peptides with amino acid sequences patterned after the autophosphorylation site in Type II regulatory subunit of the cAMP-dependent protein kinase. The phosphatase was activated by Ni2+ and Mn2+, and stimulated further by calmodulin. In the presence of Ni2+ and calmodulin, it exhibited similar kinetic constants for the dephosphorylation of phosphotyrosyl LC20 (Km = 0.9 microM, and Vmax = 350 nmol/min/mg) and phosphoseryl LC20 (Km = 2.6 microM, Vmax = 690 nmol/min/mg). Dephosphorylation of phosphotyrosyl LC20 was inhibited by phosphoseryl LC20 with an apparent Ki of 2 microM. Compared to the reactions with phosphotyrosyl LC20 as the substrate, reactions with phosphotyrosine-containing oligopeptides exhibited slightly higher Km and lower Vmax values. The reaction with the phosphoseryl peptide based on the Type II regulatory subunit sequence exhibited a slightly higher Km (23 microM), but a much higher Vmax (4400 nmol/min/mg) than that with its phosphotyrosine-containing counterpart. Micromolar concentrations of Zn2+ inhibited the phosphatase activity; vanadate was less potent, and 25 mM NaF was ineffective. The study provides quantitative data to serve as a basis for comparing the ability of the calmodulin-dependent protein phosphatase to act on phosphotyrosine- and phosphoserine-containing substrates.     

The use of cobalt ions as a collisional quencher to probe surface charge and stability of fluorescently labeled bilayer vesicles

Co2+ quenched the fluorescence of the lipid probes NBD-phosphatidylethanolamine (NBD-PE) and lissamine-rhodamine phosphatidylethanolamine (N-Rh-PE) incorporated into lipid vesicles, according to a collisional quenching mechanism in agreement with the Stern-Vollmer law. The quenching coefficient (Q) for NBD-PE, incorporated into uncharged phosphatidylcholine (PC) vesicles was 13.8 M-1. This value was equal to the quenching coefficient of water-soluble NBD-taurine in aqueous solution, indicating that Co2+ was readily accessible to the outer surface of PC vesicles. In phosphatidylserine-phosphatidylethanolamine (PS-PE) (1:1) vesicles, quenching was also proportional to Co2+ concentration but Q was 114 mM-1, some 8000-fold smaller. Using the Gouy-Chapman-Stern model we demonstrated that the surface density of Co2+ bound to lipid was linear with Co2+ concentration in the medium up to 7%. Co2+-associated phospholipid would in turn quench NBD-PE or N-Rh-PE by collisional quenching with lateral diffusion. We investigated the ability of Co2+ to permeate PS-PE (1:1) vesicles. Co2+ quenched fluorophores on the outer surface of large unilamellar vesicles, formed by reverse-phase evaporation. In small unilamellar vesicles Co2+ quenched probes on both outer and inner surfaces, indicating rapid permeation of the ions into the vesicles. Using stopped-flow rapid mixing, we measured the rate of influx of Co2+, and correcting for surface potential using the Gouy-Chapman-Stern model, we calculated a permeability coefficient of 10(-12) cm/s for Co2+ concentrations below 300 microM. Above this concentration, there was a very steep rise in the permeability coefficient, indicating that binding of Co2+ induces defects in the bilayer of these vesicles. This may be related to the ability of the vesicles to undergo membrane fusion. A method for calculating the membrane surface potential from Co2+ quenching data is presented.     

The nucleotide sequence of a nematode vitellogenin gene.

The nematode, Caenorhabditis elegans, contains a family of six genes that code for vitellogenins. Here we report the complete nucleotide sequence of one of these genes, vit-5. The gene specifies a mRNA of 4869 nucleotides, including untranslated regions of 9 bases at the 5' end and 51 bases at the 3' end. Vit-5 contains four short introns totalling 218 bp. The predicted vitellogenin, yp170A, has a molecular weight of 186,430. At its N terminus it is clearly related to the vitellogenins of vertebrates. However, the vit-5-encoded protein does not contain a serine-rich sequence related to the vertebrate vitellin, phosvitin. In fact, the amino acid composition of the nematode protein is very similar to that of the vertebrate protein without phosvitin. Vit-5 has a highly asymmetric codon choice dictionary. The favored codons are different from those favored in other organisms, but are characteristic of highly expressed C. elegans genes. The strong selection against rare codons is not as great near the 5' end of the gene; rare codons are 15 times more frequent within the first 54 bp than in the next 4.8 kb.     

The folding of ovalbumin. Renaturation in vitro versus biosynthesis in vitro.

Hen ovalbumin, the major secretory product of oviduct cells, is a 43 000-dalton glycoprotein. Many studies have led to controversy over the question of whether ovalbumin (OA) can be fully renatured after chemical denaturation. We have studied the renaturation of OA after denaturation with guanidinium chloride, urea or alkaline pH. Denatured OA displays an intrinsic viscosity consistent with nearly complete unfolding of the protein. Removal of the denaturant results in a complete reversal of the changes in intrinsic viscosity. However, closer examination of the renatured protein reveals major differences from the native form. Renatured OA (OAR) can be completely separated from the native form (OAN) by affinity chromatography on phenyl-Sepharose. OAR displays altered tryptophan fluorescence, u.v.-absorption and c.d. spectra. Only OAR binds anilinonaphthalenesulphonate (as measured by fluorescence enhancement). OAR, but not OAN, binds about 2 mol of the covalent hydrophobic affinity probe phenyl isothiocyanate/mol. Renaturation, and the production of OAR, occurs regardless of the oxidation state of the disulphide bonds, of phosphorylation of the protein, and of the presence or the absence of the single carbohydrate chain. OAR may be either monomeric or an irreversible aggregate. Which of these two states is formed depends on the protein concentration during renaturation. Monomeric and aggregated OAR can be distinguished on the basis of some spectroscopic characteristics, but they share the essential hydrophobic characteristics that distinguish them from OAN. OAN and OAR do not spontaneously interconvert. Antibodies raised to each can be made monospecific by immunoabsorption. Thus two stable forms of OA can be obtained, one of which, OAR, displays hydrophobic characteristics. OAN, but not OAR, is formed when OA is synthesized in vitro in a translation system.     

DETERMINATION OF GUSTATORY SUCROSE THRESHOLD IN WATER BY USE OF A LINEAR SUCROSE GRADIENT

A novel experimental method was developed which allows the determination of the threshold concentration of sucrose by use of a linear sucrose gradient in water. With this method a continuous tasting of the test-liquid is possible. A panel of 15 persons experienced in taste-testing was used. Three gradients of different steepness were applied: 0 to 1.5% (w/w) sucrose in 2 min (I), 3 min (II) and 4 min (III). The results of the new method were compared with those of the standard method (DIN). With gradients I and II we found values which were significantly higher than those of the standard method (I: 0.49% (w/w); II: 0.46% (w/w); DIN: 0.31% (w/w)), whereas with gradient III the same threshold value was found as with the DIN-Method (III: 0.32% (w/w)).