Nitrite Transport in Chloroplast Inner Envelope Vesicles (I. Direct Measurement of Proton-Linked Transport)

Chloroplast inner envelope membrane vesicles that are loaded with the pH-sensitive fluorophore, pyranine, show rapid internal acidification when nitrite is added. Acidification is dependent upon [delta]pH, with the inside of vesicles being alkaline with respect to the outside. The rate of vesicle acidification was directly proportional to the concentration of nitrite that was added and the imposed pH difference across the membrane. In contrast, added nitrate had no effect on vesicle acidification. Nitrite also caused acidification of asolectin vesicles. The extent of vesicle acidification is dependent on the internal volume of vesicles. Inner envelope and asolectin vesicles that were prepared by extrusion were approximately the same size, allowing them to be compared when the final extent of acidification, measured after the pH gradient had collapsed, was similar. The rate of nitrite-dependent acidification was similar in these two preparations at any single nitrite concentration. These results indicate that nitrite movement occurs by rapid diffusion across membranes as nitrous acid, and this movement is dependent on a proton gradient across the lipid bilayer. Under conditions approximating those in vivo, the rate of diffusion of nitrous acid far exceeds that of nitrite reduction within chloroplasts.     

Molecular evolution of mitochondrial 12S RNA and cytochrome b sequences in the pantherine lineage of Felidae

DNA sequence comparisons of two mitochondrial DNA genes were used to infer phylogenetic relationships among 17 Felidae species, notably 15 in the previously described pantherine lineage. The polymerase chain reaction (PCR) was used to generate sequences of 358 base pairs of the mitochondrial 12S RNA gene and 289 base pairs of the cytochrome b protein coding gene. DNA sequences were compared within and between 17 felid and five nonfelid carnivore species. Evolutionary trees were constructed using phenetic, cladistic, and maximum likelihood algorithms. The combined results suggested several phylogenetic relationships including (1) the recognition of a recently evolved monophyletic genus Panthera consisting of Panthera leo, P. pardus, P. onca, P. uncia, P. tigris, and Neofelis nebulosa; (2) the recent common ancestry of Acinonyx jubatus, the African cheetah, and Puma concolor, the American puma; and (3) two golden cat species, Profelis temmincki and Profelis aurata, are not sister species, and the latter is strongly associated with Caracal caracal. These data add to the growing database of vertebrate mtDNA sequences and, given the relatively recent divergence among the felids represented here (1-10 Myr), allow 12S and cytochrome b sequence evolution to be addressed over a time scale different from those addressed in most work on vertebrate mtDNA.      

A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates.     

A Similar Dichotomy of Sugar Modulation and Developmental Expression Affects Both Paths of Sucrose Metabolism: Evidence from a Maize Invertase Gene Family

Invertase and sucrose synthase catalyze the two known paths for the first step in carbon use by sucrose-importing plant cells. The hypothesis that sugar-modulated expression of these genes could provide a means of import adjustment was initially suggested based on data from sucrose synthases alone; however, this hypothesis remained largely conjectural without critical evidence for invertases. Toward this end, a family of maize invertases was cloned and characterized. Here, we show that invertases are indeed sugar modulated and, surprisingly, like the sucrose synthase genes, fall into two classes with contrasting sugar responses. In both families, one class of genes is upregulated by increasing carbohydrate supply (Sucrose synthase1 [Sus1] and Invertase2 [Ivr2]), whereas a second class in the same family is repressed by sugars and upregulated by depletion of this resource (Shrunken1 [Sh1] and Invertase1 [Ivr1]). The two classes also display differential expression during development, with sugar-enhanced genes (Sus1 and Ivr2) expressed in many importing organs and sugar-repressed, starvation-tolerant genes (Sh1 and Ivr1) upregulated primarily during reproductive development. Both the Ivr1 and Ivr2 invertase mRNAs are abundant in root tips, very young kernels, silk, anthers, and pollen, where a close relationship is evident between changes in message abundance and soluble invertase activity. During development, patterns of expression shift as assimilate partitioning changes from elongating silks to newly fertilized kernels. Together, the data support a model for integrating expression of genes differentially responsive to carbohydrate availability (i.e., feast and famine conditions) with developmental signals. The demonstration that similar regulatory patterns occur in both paths of sucrose metabolism indicates a potential to influence profoundly the adjustment of carbon resource allocation.     

Sea urchin Hox genes: insights into the ancestral Hox cluster

We describe the Hox cluster in the radially symmetric sea urchin and compare our findings to what is known from clusters in bilaterally symmetric animals. Several Hox genes from the direct-developing sea urchin Heliocidaris erythrogramma are described. CHEF gel analysis shows that the Hox genes are clustered on a < or = 300 kilobase (kb) fragment of DNA, and only a single cluster is present, as in lower chordates and other nonvertebrate metazoans. Phylogenetic analyses of sea urchin, amphioxus, Drosophila, and selected vertebrate Hox genes confirm that the H. erythrogramma genes, and others previously cloned from other sea urchins, belong to anterior, central, and posterior groups. Despite their radial body plan and lack of cephalization, echinoderms retain at least one of the anterior group Hox genes, an orthologue of Hox3. The structure of the echinoderm Hox cluster suggests that the ancestral deuterostome had a Hox cluster more similar to the current chordate cluster than was expected Sea urchins have at least three Abd-B type genes, suggesting that Abd-B expansion began before the radiation of deuterostomes.      

Reduced product formation following perturbation of ethanol- and propionate-fed methanogenic CSTRs

Energetic analysis was applied to reduced product formation following perturbation of ethanol- and propionate-fed methanogenic continuous stirred tank reactors (CSTRs). Formation and dissipation of longer-chained n-carboxylic acids corresponded with the variation in Gibbs free energy change associated with beta-oxidation reactions. Formation appeared to occur from acetate and propionate by reductive back-reactions, made energetically favorable by elevated hydrogen partial pressure (P(H(2) )), and possibly mediated by biosynthetic enzymes. The formed longer-chained acids dissipated when the P(H(2) ) fell and equilibrium shifted to favor beta-oxidations. n-Propanol was found to be produced from propionate in a coupled ethanol oxidation/propionate reduction reaction, mediated by ethanol-oxidizing organisms during high rates of ethanol utilization and elevated P(H(2) ). When P(H(2) ) declined, n-propanol was oxidized back to its precursor propionate. Both reaction energetics and intracellular diffusion of the electron carrier may effect transient mediation of this coupled reaction.