Thermodynamic parameters are sequence-dependent for the supercoil-induced B to Z transition in recombinant plasmids

The entropy and enthalpy changes which contribute to the thermodynamics of the B to Z transition were determined for three recombinant plasmids containing a (dC-dG)16 tract and for a plasmid containing a pair of (dT-dG)20 regions. For each base pair which adopts a left-handed conformation in the plasmids with (dC-dG)16 sequences, the delta HBZ and delta SBZ are -2.1 kcal/mol bp and -8.8 cal/K-mol bp, respectively. In the plasmid containing the (dT-dG)20 tracts, however, the delta HBZ and delta SBZ values are 0.58 kcal/mol bp and -0.76 cal/K-mol bp, respectively. Also, these determinations show that for each B-Z junction that forms in the plasmids containing the (dC-dG), the enthalpy and entropy changes are 24 kcal/mol junction and 65 cal/K-mol junction, whereas for the (dT-dG) plasmid, the enthalpy and entropy changes are -1.8 kcal/mol junction and -22 cal/K-mol junction, respectively. Those values for the enthalpy and entropy changes for the formation of a BZ junction in (dC-dG) and (dT-dG) plasmids suggest that the properties and possibly the structures of the junctions are different. Calculations using the enthalpy and entropy changes determined in this study reveal that the B to Z transition in plasmids containing (dC-dG) blocks are more temperature-dependent than the transitions in plasmids with (dT-dG) blocks. Surprisingly, at temperatures above 60 degrees C, calculations indicate that the B to Z transitions in (dT-dG) plasmids should be energetically favored over that transition in (dC-dG) plasmids.     

Development,Labour Relations and Gender in Papua New Guinea.

This paper examines the relationship between ‘subsistence’ production, simple commodity production and wage labour and the different effects this relationship has on males and females. The peri-urban village of Siar, located a few kilometres north of Madang town in Papua New Guinea, is used as a case study. It is argued that the village as a social group is dependent on wage labour for its reproduction and hence is proletarianized. As part of the proletarianization process, married women in the village have become doubly subordinated: to capital and to men.     

Vesicular stomatitis virus-infected cells fuse when the intracellular pool of functional M protein is reduced in the presence of G protein.

Five highly cytolytic strains of both Indiana and New Jersey serotypes of vesicular stomatitis virus were shown to induce cell fusion in BHK-21 and R(B77) cells. Inhibition of protein synthesis after the eclipse period of viral replication is a prerequisite for vesicular stomatitis virus-induced cell fusion. Pulse-chase experiments showed that inhibition of protein synthesis would lead to a drastic reduction in the intracellular pool of M protein as compared with other proteins. A temperature-sensitive mutant defective in M protein function (G31) was the only mutant of the five complementation groups to spontaneously induce polykaryocytes at the nonpermissive temperature. Previously, G protein has been shown to play a role in vesicular stomatitis virus-induced cell fusion. These results suggest that the combination of the presence of G protein on the virus-infected cell surface and the absence of functional M protein or a reduced level of intracellular M protein promotes cell fusion. On the basis of this study, we propose that vesicular stomatitis virus infection can induce cell fusion when the functional M protein pool declines to a critical level while G protein remains on the cell surface.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Isolation of chitin synthetase from Saccharomyces cerevisiae. Purification of an enzyme by entrapment in the reaction product

Chitin synthetase, in the zymogen form, was extracted with digitonin from a particulate fraction from Saccharomyces cerevisiae and converted into active form by treatment with immobilized trypsin. When the activated enzyme was incubated with UDP-GlcNAc and other components of an assay mixture, a chitin precipitate formed, trapping a large portion of the synthetase. The enzyme was easily extracted frm the chitin gel with a recovery of approximately 50% and an enrichment of approximately 100-fold. Further purification was obtained by repeating the chitin step. After polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the purified synthetase showed a major band corresponding to Mr 63,000, a weaker band at Mr 74,000, and some other minor bands. Under nondenaturing conditions, an Mr of 570,000 was calculated for the enzyme from Stokes radius and sedimentation coefficient determinations. After electrophoresis in a nondenaturing gel and incubation with the components of the standard assay, chitin was formed and precipitated in the gel, yielding an opaque band. Soluble oligosaccharides were not precursors for insoluble chitin, suggesting that synthesis of chitin chains takes place by a processive mechanism. N-Acetylglucosamine stimulated the purified synthetase only slightly and did not participate as a primer in the reaction. The same chain length, somewhat more than 100 units of GlcNAc, was determined in samples of chitin that had been synthesized either in vivo, or with a membrane preparation or with purified synthetase. These results suggest that chitin synthetase itself is capable both of initiating chitin chains without a primer and of determining their length.     

Structural characterization of virion proteins and genomic RNA of human parainfluenza virus 3

The virion proteins and genomic RNA of human parainfluenza virus 3 have been characterized. The virion contains seven major and two minor proteins. Three proteins of 195 X 10(3) molecular weight (195K), 87K, and 67K are associated with the nucleocapsid of the virion and have been designated L, P, and NP, respectively. Three proteins can be labeled with [14C]glucosamine and have molecular weights of 69K, 60K, and 46K. We have designated these proteins as HN, F0, and F1, respectively. HN protein has interchain disulfide bonds, but does not participate in disulfide bonding to form homomultimeric forms. F1 appears to be derived from a complex, F1,2, that has an electrophoretic mobility similar to that of F0 under nonreducing conditions. A protein of 35K is associated with the envelope components of the virion and aggregates under low-salt conditions; this protein has been designated M. The genome of human parainfluenza virus 3 is a linear RNA molecule with a molecular weight of approximately 4.6 X 10(6).     

Alterations in the Structure of the Oligosaccharide of Vesicular Stomatitis Virus G Protein by Swainsonine

Swainsonine, an inhibitor of glycoprotein processing, inhibits the formation of the normal oligosaccharide chain of the G protein of vesicular stomatitis virus. Thus, when vesicular stomatitis virus was grown in baby hamster kidney cells in the presence of swainsonine (15 to 500 ng/ml) and labeled with [2-(3)H]mannose, the oligosaccharide portion of the G protein was completely susceptible to the action of endoglucosaminidase H. However, the normal viral glycoprotein is not susceptible to this enzyme. Various enzymatic treatments and methylation studies of the mannose-labeled oligosaccharides suggest that swainsonine causes the formation of a hybrid-type oligosaccharide having an oligomannosyl core (Man(5)GlcNAc(2)-Asn) characteristic of neutral oligosaccharides plus the branch structure (NeuNAc-Gal-GlcNAc) characteristic of the complex oligosaccharides. A structure for this hybrid oligosaccharide is proposed. Swainsonine had no effect on the incorporation of [(14)C]leucine into viral proteins, nor did it change the number of PFU produced in these cultures. It did, however, slightly decrease the incorporation of [(3)H]glucosamine and increase the incorporation of [(3)H]mannose. Vesicular stomatitis virus raised in the presence of swainsonine bound much more tightly to columns of concanavalin A-Sepharose than did control virus. Swainsonine had to be added within the first 4 or 5 h of virus infection to be effective. Thus, when 100 ng of the alkaloid per ml was added at any time within the first 3 h of infection, essentially all of the glycoprotein was susceptible to digestion by endoglucosaminidase H. However, when swainsonine was added 4 h after the start of infection, 30% of the glycopeptides became resistant to endoglucosaminidase H; at 5 h, 70% were resistant. The effect of swainsonine was reversible since removal of the alkaloid allowed the cells to form the normal complex glycoproteins. However, the time of removal was critical in terms of oligosaccharide structure.     

Lymphocyte zinc metabolism in disease

Lymphocytes were obtained from two patients with paroxysmal nocturnal hemoglobinuria as well as from apparently healthy controls and from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. Subsequently, several aspects of zinc metabolism were studied in vitro in short-term cultures of these lymphocytes in order to assess lymphocyte functional capacity. The results of mitogen stimulation and zinc uptake studies for lymphocytes from donors with paroxysmal nocturnal hemoglobinuria were similar to those obtained for leukemic lymphocytes. The results of studies to determine the inducibility of the low molecular weight zinc-binding protein, metallothionein, by zinc were complicated by the decrease in overall protein synthesis in lymphocytes from donors in the paroxysmal nocturnal hemoglobinuria. It is proposed that paroxysmal nocturnal hemoglobinuria is indeed a clonal disorder and the relationship between lymphocytes in this disorder and leukemic lymphocytes is discussed.