Polyethylene glycol in aqueous solution: Solvent perturbation and gel filtration studies

As part of an investigation of the mechanism of precipitation of proteins by synthetic polymers, we measured the ability of oligomers and polymers of ethylene glycol to (a) alter the solubility of amino acids, (b) perturb the absorption spectra of aromatic amino acids, and (c) enhance the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS). All of these effects increased with increasing degree of polymerization, up to a molecular weight of about 400. This trend can be partially attributed to the diminishing influence of the terminal OH groups. However, small analogs such as dioxane and dimethoxyethane (lacking OH groups) were much less effective than the polymers, suggesting a cooperative effect between neighboring ethoxide residues in the polymer. In contrast, the effectiveness of polyethylene glycol (PEG) as a protein-precipitating agent continued to increase with increasing size; polymers of molecular weight 4000–6000 were substantially more effective than their 10-fold smaller homologues. On exclusion chromatographic columns, the synthetic polymers eluted much earlier than did proteins of comparable molecular weight. This discrepancy diminished in 6 m guanidine where the proteins eluted much earlier, reflecting their conversion to random coils. In contrast, the elution of PEG was only slightly affected, suggesting a random coil configuration even in the absence of denaturant. PEG-20M, a mixture prepared by coupling 2 mol of PEG-6000 with an epoxide, was resolved into two components by exclusion chromatography on Sephadex G-100. The fast-eluting component (the coupled product) was unusually effective in enhancing ANS fluorescence and was shown to bind the dye with K_a = 5.1 × 10³, M^?1 at 25 °C.     

Characterization of exoglucanase and synergistic hydrolysis of cellulose in Clostridium stercorarium

Abstract A cellobiohydrolase component was isolated from an anaerobic thermophilic cellulolytic bacterium, Clostridium stercorarium . When acting alone, the enzyme showed minimal activity towards ordered substrates such as cellulose and filter paper but it has been shown to attack phosphoric-acid swollen cellulose giving cellobiose as principal product. When recombined with endoglucanase it did allow an extensive hydrolysis demonstrating a marked synergism in the action of those two components; the addition of β-glucosidase resulted in a further increase in activity.     

Sequences homologous to Tc(s) transposable elements ofCaenorhabditis elegans are widely distributed in the phylum nematoda

Summary To have a better understanding of the evolutionary history of mobile elements within the nematodes, we examined the distribution and the conservation of homologues to transposable elements fromCaenorhabditis elegans (Tc1, Tc2, Tc3, Tc4, Tc5, and FB1) in 19 nematode species belonging to the class Secernentea. Our results show that Tc1 elements display a distribution restricted to the family Rhabditidae with poor conservation. The Tc2 and FB1 homologous elements have the same patchy distribution within the Rhabditidae. They were only found inCaenorhabditis and inTeratorhabditis. The Tc3 element is widely distributed among nematode species. Tc3 homologous elements are present in the majority of the Rhabditidae but also in two genera within the family Panagrolaimidae, and inBursaphelenchus, which belongs to the order Aphelenchida. Tc4 and Tc5 homologues show the most limited distribution of all tested elements, being strictly limited toC. elegans. These data indicate that in some cases, the distribution of transposable elements in the nematode cannot be explained by strict vertical transmission. The distribution of Tc3, Tc4, and Tc5 suggests that horizontal transmission may have occurred between reproductively isolated species during their evolutionary history.     

Evolution of mouse B1 repeats: 7SL RNA folding pattern conserved

Summary In a recent report mouse B1 genomic repeats were divided into six families representing different waves of fixation of B1 variants, consistent with the retroposition model of human Alu elements. These data are used to examine the distribution of nucleotide substitutions in individual genomic repeats with respect to family consensus sequences and to compare the minimal energy structures of the corresponding B1 RNAs. By an enzymatic approach the predicted structure of B1 RNAs is experimentally confirmed using as a model sequence an RNA of a young B1 family member transcribed in vitro by T7 RNA polymerase. B1 RNA preserves folding domains of the Alu fragment of 7SL RNA, its progenitor molecule. Our results reveal similarities among 7SL-like retroposons, human Alu, and rodent B1 repeats, and relate the evolutionary conservation of B1 family consensus sequences to selection at the RNA level.     

Domain structure and interactions of recombinant urokinase-type plasminogen activator.

Urokinase-type plasminogen activator (uPA) is a mosaic glycoprotein composed of an epidermal growth factor-like (EGF), a kringle and a serine protease (SP) module. It exists in single and two-chain forms designated HMW pro-uPA and HMW uPA, respectively. A low molecular weight form, LMW uPA, lacks the EGF and kringle modules and is composed of the SP module alone. Recombinant-expressed proteins representing both HMW forms exhibit four reversible unfolding transitions that are resolved by deconvolution of melting curves obtained by differential scanning calorimetry at pH 4.5; no differences in the melting properties of the single and two-chain forms were found. The proteolytic fragment Ser1-Lys135 (EGF-kringle) exhibits two transitions, while the isolated EGF and kringle modules each exhibit a single two-state transition. Thus, both of these modules retain an independently folded compact structure when isolated. The isolated SP module (LMW uPA) exhibits two closely spaced transitions at low pH indicating the melting of two domains of similar stability. Fluorescence-detected melting curves of LMW uPA reveal increasing cooperativity with increasing pH, suggesting an increase in the interaction between the two SP domains. Treatment of both HMW and LMW uPA with the tripeptide inhibitor Glu-Gly-Arg-chloromethylketone dramatically increased the stability of both domains of the SP module which now melt together in a single two-state transition, even at low pH, with no effect on the EGF and kringle modules. From these data one concludes that UK consists of four independently folded domains. Two are formed by the EGF and kringle modules which do not interact with each other or with the SP module. The SP module contains two domains that are independent at low pH but exhibit a tendency to merge into a single cooperative unit at neutral pH or after treatment with the tripeptide inhibitor.     

Dynamics of carbon assimilation by St Lawrence estuary phytoplankton at the end of summer

This study presents data of in situ measurements of inorganic carbon assimilation by phytoplankton communities of the St Lawrence estuary during the end of summer 1982. We used carboxylase activity measurements (ribulose-1,5-bisphosphate carboxylase, carboxylases) and the ¹³C/¹²C ratio of phytoplankton organic carbon, expressed as ¹³C, to study patterns of assimilation. Upper estuary phytoplankton communities showed a smaller turn-over rate in carbon assimilation than lower estuary phytoplankton communities. Carbon assimilation was limited by light intensity in the upper estuary and by CO₂ availability in the lower estuary. In the St Lawrence estuary, stable carbon isotope ratios of phytoplankton organic carbon seemed to be controlled by inorganic carbon availability rather than by phytoplankton metabolism.     

Fungal Catabolism of Crown Gall Opines

This study was conducted to determine the capacities of 37 fungi to utilize various crown gall opines as their sole carbon and nitrogen source. One strain of Fusarium solani, two of Cylindrocarpon destructans, and six of Cylindrocarpon heteronema catabolized octopine, mannopine, octopinic acid, succinamopine, or a combination of these opines. One C. heteronema and one Fusarium dimerum strain grew only on succinamopine. None of the fungal isolates had the ability to grow on nopaline. The catabolism of opines by fungi was confirmed by the disappearance of the opine from the growth medium and by an increase in final mycelial dry weight with rising initial concentration of test substrate. This study thus shows that the catabolism of opines is not restricted to bacteria.     

Patterns of chromosome segregation in chinese hamster × mouse cell hybrids between permanent cell lines and thymus cells

Hybrids between a tumorigenic Chinese hamster cell line (DC3F-aza) and normal mouse thymus cells very rapidly lost most of their mouse chromosomes, whereas hybrids between tumorigenic mouse cell lines (either Cl.1D of L cell line origin, or PCC4-aza1 teratocarcinoma cells) and normal Chinese hamster thymus cells lost most of their hamster chromosomes. From three such fusion experiments, 20 cell lines were developed which all followed the same evolution, namely, the elimination of the majority of the chromosomes contributed by the normal thymus cell. In some hybrids, the elimination process resulted in the total absence of intact chromosomes contributed by the thymus cell parent. Such hybrids were distinguished from revertant parental cells growing in the selective hybrids were distinguished from revertant parental cells growing in the selective medium by the presence of at least one enzyme in their cell extracts which displayed the electrophoretic mobility of the enzyme of the thymus cell parent. These observations, together with data from other reports, suggest that, as a rule, interspecific cell hybrids which develop upon fusion between normal diploid cells and tumorigenic cell lines maintain the chromosomes of the latter and eliminate preferentially many or most of the chromosomes contributed by the normal cell parents, independent of the respective species of the parental cells.     

Mapping of the mitochondrial 16S ribosomal RNA gene and its expression in the cytoplasmic petite mutants ofSaccharomyces cerevisiae

The 16S ribosomal RNA gene of yeast mitochondria was titrated in various cytoplasmic petite mutants by DNA-RNA hybridization. The gene was located close to the prolyl transfer RNA gene. The properties of the rho^? strains suggest that the gene order would be: - P_I - 16S - prolyl tRNA - valyl tRNA - (tRNAs) - R_I - R_III -; the 23S ribosomal gene is far from the 16S one. Several petite mutants were found which have retained, in addition to many transfer RNA genes, both of the 23S and 16S ribosomal RNA genes. The two genes seem to be transcribed in these mutants.