Control of leghaemoglobin synthesis in snake beans

1. The finding that the plant is the genetic determinant of leghaemoglobin production in legume nodules was further tested by inoculating snake beans with two strains of Rhizobium selected to give large genetic differences. Carbohydrate requirement patterns, immunological techniques and DNA base ratio determinations were used to demonstrate genetic differences between the two rhizobial strains. 2. Partially purified preparations of the haemoglobins from the nodules produced by the two strains showed no differences when examined by electrophoresis, isoelectric focusing or ion-exchange chromatography. 3. Two different leghaemoglobins from each type of nodule were separated by chromatography on DEAE-cellulose. One of these was isolated in the Fe(3+) form and accounted for two-thirds of the total leghaemoglobin. When it was examined in the analytical ultracentrifuge and by amino acid analysis, this major component did not vary with the inoculant rhizobial strain. The molecule had an s(20,w) of 1.88S, a diffusion coefficient of 10.7x10(-7)cm(2).s(-1) and a mol. wt. of 16700. 4. These results strongly support the hypothesis that the mRNA for leghaemoglobin is transcribed from plant DNA.     

Effect of sulfur supply on sulfate uptake,and alkaline sulfatase activity in free-living and symbiotic bradyrhizobia

The effect of sulfur limitation on sulfate transport and metabolism was studied in four bradyrhizobia strains using sulfur-limited and sulfur-excess chemostat cultures. Characteristics of bradyrhizobia associated with sulfurlimitation were determined and these parameters used to bioassay the sulfur status of bacteroids in nodules on sulfur adequate or sulfur deficient soybean and peanut plants. Sulfur-limited cells took up sulfate 16- to 100-fold faster than sulfur-rich cells. The sulfate-uptake system appeared similar in all strains with apparent K _m values ranging from 3.1 M to 20 M sulfate with maximum activities between 1.6 and 10 nmol·min^-1·mg^-1 protein of cells. Sulfate-limited cells of all strains derepressed the enzyme alkaline sulfatase in parallel with the derepression of the sulfate transport system. Similarly, the initial enzyme of sulfate assimilation (ATP sulfurylase) was fully derepressed in sulfur-limited cultures. Bacteroids isolated from sulfur adequate and sulfur deficient soybean and peanut possessed very limited sulfate uptake activity and low levels of activity of ATP sulfurylase as well as lacking alkaline sulfatase activity. These results indicate bacteriods have access to adequate sulfur to meet their requirements even when the host plant is sulfur-deficient.Abbreviations CCCP Carbonyl cyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide     

Chromosome replication in cell-free systems from Xenopus eggs

Cell-free systems from eggs of the frog Xenopus laevis are able to perform most of the acts of eukaryotic chromosome replication in vitro. This now includes the crucial regulatory step of initiation, which had only been achieved for viral systems previously. Purified DNA or nuclei are able to initiate and complete semi-conservation replication in egg extracts in vitro (Blow & Laskey, Cell 47, 557-587 (1986). Replication does not require specialized DNA sequences either in vitro or in microinjected eggs, but in both systems large templates replicate more efficiently than small templates. In some cases replication can re-initiate, excluding the possibility that replication is primed by preexisting primers in the template preparations. When nuclei are replicated in vitro, only one round of replication is observed in a single incubation resembling the single round of replication observed for purified DNA after micro-injection. The mechanism that prevents re-initiation of replication within a single cell cycle is discussed and certain models are eliminated. Nucleosome assembly from histones and DNA has also been studied in cell-free systems from Xenopus eggs. Fractionation has led to the identification of two acidic proteins called nucleoplasmin and N1, which bind histones and transfer them to DNA. The sequences of both proteins have been determined by cDNA cloning and sequencing. Both proteins are found as complexes with histones in eggs.     

Proteomic and genomic characterization of chromatin complexes at a boundary

We have dissected specialized assemblies on the Saccharomyces cerevisiae genome that help define and preserve the boundaries that separate silent and active chromatin. These assemblies contain characteristic stretches of DNA that flank particular regions of silent chromatin, as well as five distinctively modified histones and a set of protein complexes. The complexes consist of at least 15 chromatin-associated proteins, including DNA pol epsilon, the Isw2-Itc1 and Top2 chromatin remodeling proteins, the Sas3-Spt16 chromatin modifying complex, and Yta7, a bromodomain-containing AAA ATPase. We show that these complexes are important for the faithful maintenance of an established boundary, as disruption of the complexes results in specific, anomalous alterations of the silent and active epigenetic states.     

Preliminary evaluation of non-native rainbow trout (Oncorhynchus mykiss) impact on the Cederberg ghost frog (Heleophryne depressa) in South Africa’s Cape Fold Ecoregion

We evaluated the impact of non-native rainbow trout Oncorhynchus mykiss on a population of endemic Cedarberg ghost frog Heleophryne depressa in the upper Krom River (Olifants-Doring River Catchment, Cape Fold Ecoregion). We compared H. depressa abundance (using kick-sampling and underwater video analysis) and environmental conditions between sites above and below a waterfall that marks the upper distribution limit of O. mykiss. Heleophryne depressa abundance was significantly greater above the waterfall than that below it, and, because there was no significant difference in measured environmental variables, O. mykiss presence is identified as the most likely explanation for the observed decrease in H. depressa abundance.     

The life of root nodule bacteria in the acidic underground

Abstract The effects of low pH on the growth and survival of root nodule bacteria are important in determining the success of legume inoculation in practical agriculture. The mechanisms for maintenance of intracellular pH (pH_i) are crucial, but little is yet known about them in root nodule bacteria. Like other Gram-negative bacteria, root nodule bacteria show an adaptive acid tolerance response, with growth at moderately acidic conditions protecting against an extreme acid shock. Variations in acid tolerance within species of root nodule bacteria imply a genetic basis to low pH tolerance and studies of acid-sensitive mutants suggest that as many as 20 genes could be involved. A general model for acid tolerance in root nodule bacteria is presented.     

Maintenance of Intracellular pH and Acid Tolerance in Rhizobium meliloti

The development and function of the Rhizobium meliloti-Medicago sp. symbiosis are sensitive to soil acidity. Physiological criteria that can be measured in culture which serve to predict acid tolerance in soil would be valuable. The intracellular pH of R. meliloti was measured using either radioactively labeled weak acids (5,5-dimethyloxazolidine-2,4-dione and butyric acid) or pH-sensitive fluorescent compounds; both methods gave similar values. Six acid-tolerant strains (WSM419, WSM533, WSM539, WSM540, WSM852, and WSM870) maintained an alkaline intracellular pH when the external pH was between 5.6 and 7.2. In contrast, two Australian commercial inoculant strains (CC169 and U45) and four acid-sensitive strains from alkaline soils in Iraq (WSM244, WSM301, WSM365, and WSM367) maintained an alkaline intracellular pH when the external pH was ≥6.5, but had intracellular pH values of ≤6.8 when the external pH was ≤6.0. Four transposon Tn5-induced mutants of acid-tolerant strain WSM419, impaired in their ability to grow at pH 5.6, showed limited control over the intracellular pH. The ability to generate a large pH gradient under acid conditions may be a better indicator of acid tolerance in R. meliloti under field conditions than is growth on acidic agar plates.     

The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane.

1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed.     

Advances in the Conceptualization and Measurement of Health Care Empowerment: Development and Validation of the Health Care Empowerment Inventory

The Health Care Empowerment Model offers direction for the investigation of patient-controlled engagement and involvement in health care. At the core of the model is the construct of Health Care Empowerment (HCE), for which there exist no validated measures. A set of 27 candidate self-report survey items was constructed to capture five hypothesized inter-related facets of HCE (informed, engaged, committed, collaborative, and tolerant of uncertainty). The full item set was administered to 644 HIV-infected persons enrolled in three ongoing research studies. Exploratory and confirmatory factor analyses resulted in a two factor solution comprising four items each on two subscales: (1) HCE: Informed, Committed, Collaborative, and Engaged HCE ICCE) and (2) HCE Tolerance of Uncertainty (HCE TU). Subscale scores were evaluated for relationships with relevant constructs measured in the three studies, including depression, provider relationships, medication adherence, and HIV-1 viral load. Findings suggest the utility of this 8-item Health Care Empowerment Inventory (HCEI) in efforts to measure, understand, and track changes in the ways in which individuals engage in health care.