Spatial Genetic Structure of the Abundant and Widespread Peatmoss Sphagnum magellanicum Brid.

Spore-producing organisms have small dispersal units enabling them to become widespread across continents. However, barriers to gene flow and cryptic speciation may exist. The common, haploid peatmoss Sphagnum magellanicum occurs in both the Northern and Southern hemisphere, and is commonly used as a model in studies of peatland ecology and peatmoss physiology. Even though it will likely act as a rich source in functional genomics studies in years to come, surprisingly little is known about levels of genetic variability and structuring in this species. Here, we assess for the first time how genetic variation in S. magellanicum is spatially structured across its full distribution range (Northern Hemisphere and South America). The morphologically similar species S. alaskense was included for comparison. In total, 195 plants were genotyped at 15 microsatellite loci. Sequences from two plastid loci (trnG and trnL) were obtained from 30 samples. Our results show that S. alaskense and almost all plants of S. magellanicum in the northern Pacific area are diploids and share the same gene pool. Haploid plants occur in South America, Europe, eastern North America, western North America, and southern Asia, and five genetically differentiated groups with different distribution ranges were found. Our results indicate that S. magellanicum consists of several distinct genetic groups, seemingly with little or no gene flow among them. Noteworthy, the geographical separation of diploids and haploids is strikingly similar to patterns found within other haploid Sphagnum species spanning the Northern Hemisphere. Our results confirm a genetic division between the Beringian and the Atlantic that seems to be a general pattern in Sphagnum taxa. The pattern of strong genetic population structuring throughout the distribution range of morphologically similar plants need to be considered in future functional genomic studies of S. magellanicum.     

Nicotinamide mononucleotide adenylyltransferase. Molecular and enzymatic properties of the homogeneous enzyme from baker's yeast

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from baker's yeast crude extract. The purification procedure is relatively simple and consists of high-salt extraction of enzyme activity and precipitation with poly(ethylenimine), followed by ion-exchange and dye ligand chromatography separations. The final enzyme preparation is homogeneous as judged by a single Coomassie blue stainable band when run on nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 200 000, calculated by gel filtration and sucrose gradient centrifugation. The protein possesses quaternary structure and is composed of four apparently identical Mr 50 000 subunits. The absorption spectrum shows a maximum at 280 nm and a minimum at 253 nm. The isoelectric point is 6.2. Amino acid composition analysis shows the presence of 28 half-cystine residues. The same result has been obtained by titrating the enzyme in denaturating conditions with Ellman's reagent after incubation with sodium borohydride. NMN adenylyltransferase is a glycoprotein containing 2% sugar, 2 mol of alkali-labile phosphate per mole of enzyme, and 1 mol of adenine moiety per mole of enzyme. Therefore, the possibility that the enzyme is ADP-ribosylated exists. The Km values for ATP, NMN, and nicotinate mononucleotide are 0.11 mM, 0.19 nM, and 5 mM, respectively. Kinetic analysis reveals a behavior that is consistent with an ordered sequential Bi-Bi mechanism. The pH optimum is in the range 7.2-8.4.     

Reexamination of the activation of yeast proteinase B at pH 5: loss of inhibition effect of proteinase B inhibitors

The activation of yeast proteinase B at pH 5 has been suggested to be due to the degradation of a specific inhibitor for the enzyme, IB, by proteinase A. However, we found that when pepstatin, which completely inhibits proteinase A, was included in the pH 5 activation mixture, the same time-dependent activation of proteinase B was observed. Furthermore, proteinase B preparations that were void of proteinase A activity were still activated by incubation at pH 5. We found that the activation of proteinase B at pH 5 was due primarily to the irreversible loss of inhibitory effect of IB, which can be resolved by isoelectrofocusing into four distinct bands with isoelectric points of 4.6, 6.1, 6.8 and 7.6. These four forms of IB showed varying degrees of stability at pH 5, which may explain some of the differing observations reported in the past.     

NAD biosynthesis in human placenta: purification and characterization of homogeneous NMN adenylyltransferase.

Nicotinamide mononucleotide (NMN) adenylyltransferase has been purified to homogeneity from human placenta. The purification procedure consists of several chromatographic steps, including dye-ligand, adsorption, and hydrophobic interaction chromatography. The final enzyme preparation is homogeneous as judged by a single silver stainable band on both nondenaturating and denaturating polyacrylamide gels. The native enzyme shows a molecular weight of about 132,000, as determined by gel filtration on a Superose 12 HR 10/30 fast protein liquid chromatography column. The protein possesses a quaternary structure and is composed of four apparently identical M(r) 33,000 subunits. Isoelectrofocusing experiments give multiple pI values ranging from pH 4.7 to 6.6. Optimum pH study shows a plateau extending from pH 6.0 to pH 9.0. Km values for NMN, ATP, NAD+, and PPi are 38, 23, 67, and 125 microM, respectively. Kinetic analysis reveals a behavior consistent with an ordered sequential Bi-Bi mechanism. Among several metabolites tested only ADP-ribose and beta-NMNH were found to significantly inhibit the enzyme activity.     

Effect of mutation in the aromatic amino acid pathway on sporulation of Saccharomyces cerevisiae.

Mutations in ARO1 and ARO2 genes coding for enzymes involved in the common part of the aromatic amino acid pathway completely block the sporulation of Saccharomyces cerevisiae when in a homozygous state, whereas mutations in all the other genes of the same pathway do not. This effect is not due to the lack of any intermediate metabolite but rather to the accumulation of a metabolite preceding chorismic acid. Shikimic acid or one of its precursors was identified as the possible inhibitor. The presence of the three aromatic amino acids in the sporulation medium restores the ability to undergo meiosis. This seems not to be due to a feedback inhibition of the first enzymes of the pathway but rather to a competition between aromatic amino acids and the inhibitor on a site specific for the meiotic process. The inhibition of sporulation seems to occur at a very early step in meiosis, as indicated by the lack of premeiotic DNA synthesis in aro1 and aro2 mutants.     

Inactivation of uridine nucleosidase in yeast. Purification and properties of an inactivating protein.

It has been previously demonstrated in our laboratory that uridine nucleosidase (EC 3.2.2.3) is subjected in yeast to inactivation. An inactivating fraction has been isolated and purified to homogeneity with a procedure which includes gel filtration, adsorption chromatography, and electrofocusing techniques. The molecular weight of the enzyme, estimated either by sodium dodecyl sulfate disc gel electrophoresis or by gel filtration is approximately 44,000. No quaternary structure was evidenced. The inactivating activity possesses proteolytic activity against casein and hemoglobin with pH optima of 2.5 and 3.2, respectively. The optimal pH for uridine nucleosidase inactivation is around 4.7. The inactivating activity as well as the proteolytic activity of the preparation can be inhibited by IA but not by IB2 and IC, yeast macromolecular inhibitors for proteinase A (EC 3.4.23.8), B (EC 3.4.22.9), and C (EC 3.4.12.8), respectively. The apparent isoelectric point is pH 4.03. The carbohydrate content is 8.5%. A comparison of the properties of the inactivating protein with those of known yeast proteinases leads to the conclusion that it is identical with the enzyme previously designated as proteinase A, which for the first time has been obtained homogeneous and characterized. It has been shown that proteinase A could play a physiological role in the uridine nucleosidase inactivation process when it is associated, as a complex, with proteinase B.     

Ionic events induced by epidermal growth factor. Evidence that hyperpolarization and stimulated cation influx play a role in the stimulation of cell growth.

Charybdotoxin, a blocker of K+ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca2+ channels, were employed in mouse NIH-3T3 fibroblasts overexpressing the epidermal growth factor (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell growth. The [Ca2+]i response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (greater than 3 x 10(-8) M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na+ and Ca2+. Pretreatment with 10 microM SC38249 induced only minor changes of the intracellular Ca2+ release by EGF (the process responsible for the initial phase of the [Ca2+]i and membrane potential responses) and blocked the persistent, second phase [Ca2+]i and the hyperpolarization responses, both dependent on Ca2+ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and growth of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, growth stimulated by EGF was inhibited, moderately (-20%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca2+]i sustained by Ca2+ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis.     

Differential coupling of dopaminergic D2 receptors expressed in different cell types. Stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis in LtK- fibroblasts, hyperpolarization, and cytosolic-free Ca2+ concentration decrease in GH4C1 cells

Dopaminergic D2 receptors are widely regarded as typical inhibitory receptors, as they both inhibit adenylyl cyclase and decrease the cytosolic free Ca2+ concentration ([Ca2+]i) by activating K+ channels. A D2 receptor has recently been cloned (Bunzow, J. R., Van Tol, H. H. M., Grandy, D. K., Albert, P., Salon, J., Christie, M. D., Machida, C. A., Neve, K. A., and Civelli, O. (1988) Nature 336, 783-787) and expressed in two different cell lines, pituitary GH4C1 cells and Ltk- fibroblasts, where it has been shown to induce inhibition of adenylyl cyclase. We have investigated the additional effector systems coupled to this receptor. The responses observed in the two cells lines, which express similar levels of receptors (0.5-1 x 10(5)/cell), were surprisingly different. In GH4C1 cells D2 receptors failed to affect phosphoinositide hydrolysis and induced a decrease of [Ca2+]i. This latter effect appears to be mediated by hyperpolarization, most likely due to the activation of K+ channels. In striking contrast, in Ltk- fibroblasts the D2 receptor induced a rapid stimulation of inositol(1,4,5)-trisphosphate (+73% at 15 s) followed by the other inositol phosphates, and an immediate increase of [Ca2+]i due to both Ca2+ mobilization from internal stores and influx from the extracellular medium. In both GH4C1 and Ltk- cells, the D2 receptor response was mediated by G protein(s) sensitive to pertussis toxin. The increases of inositol trisphosphate and [Ca2+]i observed in Ltk- cells required dopamine concentrations only slightly higher than those inhibiting adenylyl cyclase (EG50 = 25, 29, and 11 nM, respectively) and were comparable in magnitude to the responses induced by the endogenous stimulatory receptor agonists, thrombin and ATP. The results demonstrate that in certain cells D2 receptors are efficiently coupled to the stimulation of phosphoinositide hydrolysis. The nature of receptor responses appears therefore to depend on the specific properties not only of the receptor molecule but also of the cell type in which it is expressed.