Characterization of Vibrio fischeri rRNA operons and subcloning of a ribosomal DNA promoter.

Analysis of rRNA genes in Vibrio fischeri indicates the presence of eight rRNA gene sets in this organism. It was found that the genes for 5S rRNA, 16S rRNA, and 23S rRNA are organized in operons in the following order: 5' end 16S rRNA 23S RNA 5S rRNA 3' end. Although the operons are homologous, they are not identical with regard to cleavage sites for various restriction endonucleases. A DNA library was constructed, and three ribosomal DNA clones were obtained. One of these clones contained an entire rRNA operon and was used as a source for subcloning. The promoter region which leads to plasmid instability was successfully subcloned into pHG165. The terminator region was subcloned into pBR322.     

Relationships among the herbicide and functional sites of acetohydroxy acid synthase from Chlorella emersonii

The properties of acetohydroxy acid synthase (AHAS, EC 4.1.3.18) from wild-type Chlorella emersonii (var. Emersonii, CCAP-211/11n) and two spontaneous sulfometuron methyl (SMM)-resistant mutants were examined. The AHAS from both mutants was resistant to SMM and cross-resistant to imazapyr (IM) and the triazolopyrimidine sulfonanilide herbicide XRD-498 (TP). The more-SMM-resistant mutant had AHAS with altered catalytic parameters (K _m, specificity), but unchanged sensitivity to the feedback inhibitors valine and leucine. The second mutant enzyme was less sensitive to the feedback inhibitors, but had otherwise unchanged kinetic parameters. Inhibition-competition experiments indicated that the three herbicides (SMM, IM, TP) bind in a mutually exclusive manner, but that valine can bind simultaneously with SMM or TP. The three herbicide classes apparently bind to closely overlapping sites. We suggest that the results with C. emersonii and other organisms can all be explained if there are separate binding sites for herbicides, feedback inhibitors and substrates.Abbreviations AHAS acetohydroxy acid synthase - AL acetolactate - AHB acetohydroxybutyrate - IM imazapyr - TP triazolopyrimidine sulfonanilide herbicide XRD-498 - R enzyme specificity - SMM sulfometuron methyl This research was supported in part by the United States — Israel Binational Science Foundation (BSF), Jerusalem, Israel (Grant 86-00205) and the Fund for Basic Research, Israel Academy of Sciences.     

Drought,heat and salt stress induce the expression of a citrus homologue of an atypical late-embryogenesis Lea5 gene

In a search for genes that are induced in citrus cell suspension in response to salt stress, a cDNA clone with high homology to cotton Lea5 gene was isolated. Data base analysis of the protein deduced from the nucleotide sequence indicates that, like in cotton, the protein from citrus contains regions with significant hydropathic character. The gene, designated C-Lea5, is expressed in citrus leaves as well as cell suspension. The steady-state level of C-Lea5 is increased in cell suspension that is grown in the presence of 0.2 M NaCl. This phenomenon is also observed in leaves of citrus plants irrigated with NaCl and in citrus seedlings which are exposed to drought and heat stress. We suggest that the osmotic stress resulted from elevated level of salt is responsible for the increase in the level of C-Lea5.     

The cyclic diguanylic acid regulatory system of cellulose synthesis in Acetobacter xylinum. Chemical synthesis and biological activity of cyclic nucleotide dimer, trimer, and phosphothioate derivatives

An unusual compound, cyclic-bis(3'----5') diguanylic acid (c-di-GMP or cGpGp), is involved in the regulation of cellulose synthesis in the bacterium Acetobacter xylinum. This cyclic dinucleotide acts as an allosteric, positive effector of cellulose synthase activity in vitro (Ka = 0.31 microM) and is inactivated via degradation by a Ca2(+)-sensitive phosphodiesterase, PDE-A (Km = 0.25 microM). A series of 13 analogs cyclic dimer and trimer nucleotides were synthesized, employing a phosphotriester approach, and tested for the ability to mimick c-di-GMP as activators of cellulose synthase and as substrates for PDE-A. Seven of the synthetic compounds stimulate cellulose synthase activity and all of these activators undergo the Ca2(+)-inhibited degradation reaction. The order of affinities for synthase activators is cGpGp approximately cdGpGp approximately cGp(S)Gp (S-diastereomer) greater than cIpGp greater than cdGpdGp greater than cXpGp greater than cIpIp greater than cGp(S)Gp (R-diastereomer). Three cyclic dinucleotides of negligible affinity for either enzyme are cApAp, cUpUp, and cCpCp. This same order of affinities essentially pertains to the analogs as inhibitors of PDE-A activity, but at least one cyclic dinucleotide, cXpXp, which does not bind to cellulose synthase, is also a substrate for the degradation reaction, demonstrating that although the two enzymes share a similar, high degree of specificity for c-di-GMP, their cyclic dinucleotide binding sites are not identical. Phosphodiester bonds of activators in which an exocyclic oxygen is replaced with an atom of sulfur (cGp(S)Gp isomers) resist the action of PDE-A, and such derivatives may be prototypes for synthetic non-hydrolyzable c-di-GMP analogs.     

Ribosomal RNA genes in Mycoplasma

Using Southern blotting analysis with labelled mycoplasmal ribosomal RNA as probe, two fragments (1 Kb and 5 Kb) were detected in an EcoR I digest of Mycoplasma capricolum DNA. This analysis revealed that the 5 Kb fragment carries both 16S rRNA sequences and the entire 23S rRNA gene of this mycoplasma. The 1 Kb fragment contains 16S rRNA sequences only. The 5 Kb EcoR I fragment has been cloned and used to characterize the structure of rRNA cistrons in various Mycoplasma strains. These experiments clearly demonstrate a substantial homology of Mycoplasma capricolum rRNA sequences with the E. coli rRNA cistron on one hand, and with Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii on the other hand. This analysis also reveals two rRNA cistrons in Mycoplasma mycoides subsp. capri and Acholeplasma laidlawii whereas one rRNA cistron is present in Mycoplasma capricolum.     

Introgressive hybridization of Senecio hercynicus and S. ovatus (Compositae,Senecioneae) along an altitudinal gradient in Harz National Park (Germany)

Introgressive hybridization of Senecio hercynicus and S. ovatus (Compositae, Senecioneae) was studied in a hybrid zone on the southern slopes of Mt Brocken (Harz Mountains, Germany). A total of 415 plants representing 10 stands along an altitudinal gradient were investigated using multivariate statistical analyses of morphological characters and molecular markers (random amplified polymorphic DNA[RAPD]). Both types of traits detected pure S. hercynicus stands on the summit plateau, pure S. ovatus stands at the lowest elevations, and hybrid swarms at intermediate elevations. While morphological and molecular patterns coincided, some individuals in hybrid stands combined morphological patterns typical of S. ovatus with RAPD patterns typical of S. hercynicus, and vice versa. In general, introgression was symmetrical within stands, though one stand combined S. ovatus characters with the glandular hair typical for S. hercynicus, and two stands combined a S. hercynicus typical RAPD genotype with morphological characters shifted towards S. ovatus. Because pure stands of S. hercynicus occurred only on the summit plateau of Mt Brocken, and markers typical for S. ovatus were detectable in stands up to 1040 m a.s.l., future fusion or assimilation of the rare form, S. hercynicus, by the more widespread S. ovatus appears possible at Mt Brocken.     

Metabolically Protective Cytokines Adiponectin and Fibroblast Growth Factor-21 Are Increased by Acute Overfeeding in Healthy Humans

Context

Circulating levels of metabolically protective and adverse cytokines are altered in obese humans and rodent models. However, it is not clear whether these cytokines are altered rapidly in response to over-nutrition, or as a later consequence of the obese state.

Methods

Forty sedentary healthy individuals were examined prior to and at 3 and 28 days of high fat overfeeding (+1250 kCal/day, 45% fat). Insulin sensitivity (hyperinsulinaemic-euglycaemic clamp), adiposity, serum levels of adiponectin and fibroblast growth factor-21 (FGF21), fatty acid binding protein-4 (FABP4), lipocalin-2 and plasminogen activator factor-1 (PAI1) were assessed. Statistics were performed by repeated measures ANOVA.

Results

Overfeeding increased weight, body fat and liver fat, fasting glucose, insulin and reduced insulin sensitivity by clamp (all P <0.05). Metabolically protective cytokines, adiponectin and FGF21 were increased at day 3 of overfeeding (P ≤0.001) and adiponectin was also elevated at day 28 (P=0.001). FABP4, lipocalin-2 and PAI-1 were not changed by overfeeding at either time point.

Conclusion

Metabolically protective cytokines, adiponectin and FGF-21, were increased by over nutrition and weight gain in healthy humans, despite increases in insulin resistance. We speculate that this was in attempt to maintain glucose homeostasis in a state of nutritional excess. PAI-I, FABP4 and lipocalin 2 were not altered by overfeeding suggesting that changes in these cytokines may be a later consequence of the obese state. Clinical trial registration: www.clinicaltrials.gov (NCT00562393)     

Activation mechanism and steady state kinetics of Bruton's tyrosine kinase

Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.     

Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor

The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.