Seedling Development and Field Performance of Prairiegrass, Grazing Bromegrass, and Orchardgrass.

Seedling establishment is a critical phase in pasture management. Knowledge of the seedling development of new forages is necessary to develop management practices and recommend species mixtures for pasture seedings. We compared seedling growth and development of prairiegrass (Bromus willdenowii Kunth = B. catharticus Vahl), grazing bromegrass (B. stamineus Desv.), and orchardgrass (Dactylis glomerata L.) in controlled environment and field studies. Seedlings were sampled weekly for 7 wk in the growth chamber and greenhouse beginning 8 to 10 d after planting (DAP). The number and mass of leaves and roots were recorded. In the field, leaf development was measured during spring and fall of 1997, and leaf and root development were measured during spring and fall of 1999. Forage dry matter (DM) yield was measured in clipped field plots during 1998 to 2000. Grazing bromegrass had more leaves, about twice the number of tillers per seedling, and a greater seedling mass than other grasses. Grazing bromegrass also had 50 to 100% more tillers m(-2) than other grasses in clipped field plots. The larger seedling size and greater tiller density, however, did not translate into greater yield in clipped plots. Grazing bromegrass yielded 10 to 15% less than orchardgrass or prairiegrass. Because of their large seedlings and rapid development, prairiegrass and grazing bromegrass probably should be used at a lower seeding rate or perhaps not used in seed mixtures with small-seeded grasses. Seedlings of these grasses should be fully established by 40 to 50 DAP under favorable moisture and temperatures in the spring and late summer.

Equilibrium and kinetic measurements reveal rapidly reversible binding of Ras to Raf.

Raf is a serine/threonine kinase that binds through its amino-terminal regulatory domain to the GTP form of Ras and thereby activates the mitogen-activated protein kinase pathway. In this study, we have characterized the interaction of the Ras-binding domain of Raf with Ras using equilibrium binding methods (scintillation proximity assay and fluorescence anisotropy), rather than with more widely used nonequilibrium procedures (such as enzyme-linked immunosorbent assay and affinity precipitation). Initial studies using glutathione S-transferase fusion proteins with either residues 1-257 or 1-190 of Raf showed that although it was possible to detect Ras binding using an enzyme-linked immunosorbent assay or affinity precipitation, it was substoichiometric; under equilibrium conditions with only a small excess of Raf almost no binding was detected. This difference was probably due to the presence of a high percentage of inactive Raf protein. Further studies used protein containing residues 51-131 of Raf, which expressed in Escherichia coli as a stable glutathione S-transferase fusion. With this protein, binding with Ras could readily be measured under equilibrium conditions. The catalytic domain of neurofibromin inhibited binding of Ras to Raf, and Raf inhibited the binding of Ras to neurofibromin showing that Raf and neurofibromin cannot be bound simultaneously to Ras. The affinities of interaction of neurofibromin and Raf with Harvey-RasLeu-61 were similar. The rate constant for dissociation of Raf from Ras was estimated to be >1 min-1, suggesting that Ras, Raf, and neurofibromin may be in rapid equilibrium in the cell. In contrast to previous reports, under equilibrium conditions there was no evidence for a difference in affinity between the minimal Ras binding domain of Raf (residues 51-131) and a region containing an additional 16 carboxyl-terminal amino acids, suggesting that residues 132-147 do not form a critical binding determinant.

Mechanism of inhibition by arachidonic acid of the catalytic activity of Ras GTPase-activating proteins.

Ras is a guanine nucleotide-binding protein that acts as a molecular switch controlling cell growth. The Ras GTPase-activating proteins (GAPs) p120-GAP and neurofibromin are candidates as Ras effectors. The GTPase-activating activity of both proteins is inhibited by mitogenic lipids, such as arachidonic acid and phosphatidic acid, and differential inhibition of the two GAPs led to the hypothesis that both were effectors in a Ras-controlled mitogenic pathway (Bollag, G., and McCormick, F. (1991) Nature 351, 576-579). We have studied the mechanism of inhibition by arachidonic acid in three ways: first, by measurements of catalytic activity under multiple turnover conditions; second, using p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid as a fluorescent probe for ligands binding to GAPs; and third, by using a scintillation proximity assay to measure direct binding of Ras to neurofibromin. We found no significant differential inhibition between p120-GAP and neurofibromin by arachidonic acid. The inhibition by arachidonic acid included a major component that is competitive with Ras GTP. These data suggest that insomuch as the mitogenic effects of lipids are mediated via inhibition of GAPs, GAPs are not Ras effector proteins. Additionally, lipids can exert a non-competitive type effect, consistent with a protein denaturing activity, making difficult extrapolations from in vitro data to the situation within cells, and possibly explaining the variability of literature data on inhibition by lipids.

Direct measurement of the binding of RAS to neurofibromin using a scintillation proximity assay.

Protein-protein interactions are of major importance in many cellular processes. When no enzymic activity is involved, assays for direct binding are required. One such example is the relatively weak interaction between oncogenic Ras and the GTPase-activating protein neurofibromin (NF1). The complex between the catalytic domain of NF1 and the GTP-form of oncogenic Ras protein dissociates rapidly; hence, equilibrium binding must be quantitated. Scintillation proximity assay (SPA) technology, a radioisotopic technique that requires no separation step, was used to characterize this interaction. Leu-61 Ras complexed with [3H]GTP was generated by nucleotide exchange in the presence of a GTP-regenerating system. A SPA signal was obtained when radiolabeled Ras was mixed with NF1 fused with glutathione S-transferase (GST), anti-GST, and protein A-coated SPA beads. This signal was abolished when any of the components were omitted and also by the addition of NaCl, which potently reduces the affinity of interaction between Ras and NF1. The neutralizing anti-Ras monoclonal antibody Y13-259 and the detergent n-dodecyl maltoside, a specific inhibitor of NF1 catalytic activity, both abolished the SPA signal from the NF1/Ras assay but neither affected a control SPA signal in which a [3H]GTP.GST-Ras fusion protein was bound to protein A-coated SPA beads. This technology could be readily extended to the measurement of other protein-protein interactions and could form the basis for high-throughput screens for the discovery of novel therapeutic agents.

Epidermal cell division and the coordination of leaf and tiller development.

Initiation and development of grass leaves and tillers are often described individually with little attention to possible interrelationships among organs. In order to better understand these interrelationships, this research examined epidermal cell division during developmental transitions at the apical meristem of tall fescue (Festuca arundinacea Schreb.). Ten seedlings were harvested each day for a 9-d period, and lengths of main shoot leaves and primary tillers were measured. In addition, numbers and lengths of epidermal cells were determined for 0.5 mm segments along the basal 3 mm of each leaf and tiller. Primordia development and onset of rapid leaf elongation were characterized by an increase in the number of cells per epidermal file with mean cell length remaining near 20 microm per cell. After the leaf had lengthened to 1-1.5 mm, cells near the leaf tip ceased dividing and increased in length, at which time leaf elongation rate increased rapidly. Ligule formation, marking the boundary between blade and sheath cells, occurred prior to leaf tip emergence above the whorl of older sheaths, while the earliest differentiation between blade and sheath cells probably began when leaves were < 1 mm long. Major transitions in leaf and tiller development appeared to be synchronized among at least three adjacent nodes. At the oldest node, cessation of cell division in the leaf sheath was accompanied by initiation of cell division and elongation in the associated tiller bud. At the next younger node the ligule was being initiated, while at the youngest node cell division commenced in the leaf primordium, as elongation of a new leaf blade began. This synchronization of events suggests a key role for the cell division process in regulating leaf and tiller development.

Kinetics of interaction between normal and proline 12 Ras and the GTPase-activating proteins, p120-GAP and neurofibromin. The significance of the intrinsic GTPase rate in determining the transforming ability of ras.

Single turnover and equilibrium binding measurements on the interaction of Gly-12 and Pro-12 Ras.GTP with the catalytic domains of the GTPase-activating proteins, p120-GAP and neurofibromin, have been made utilizing fluorescent 2'(3')O-(N-methylanthraniloyl)-nucleotides. These have enabled the equilibrium dissociation constants (Kd) for their initial binding and the rate constants of the hydrolysis step to be measured. p120-GAP binds to both Ras proteins with a Kd of 17 microM, whereas neurofibromin binds to both Ras proteins with a Kd of 1 microM. Both p120-GAP and neurofibromin increased the rate constant of the GTP hydrolysis step of Pro-12 Ras, but the maximal activation at 30 degrees C was 120-fold and 560-fold, as compared with 70,000- and 52,000-fold, with Gly-12 Ras. The affinity with which p120-GAP and neurofibromin binds to either Gly-12 or Pro-12 Ras protein was decreased dramatically by increasing ionic strength caused by addition of NaCl. The rate constant of the cleavage step of hydrolysis catalyzed by neurofibromin increases with increasing ionic strength, whereas that catalyzed by p120-GAP appears to be unaffected. The high ionic strength within the cell might result in a much lower overall GTPase-activating protein activity than is measured under conditions of low ionic strength in vitro, with p120-GAP being more severely inhibited. The GTP hydrolysis rate of Pro-12 Ras is 2-fold faster than that of normal Ras. The low oncogenicity of Pro-12 ras is explained by a model in which the intrinsic rates of hydrolysis and exchange, as well as GTPase-activating protein- and exchange factor-stimulated rates, are determinants of the biological activity of Ras proteins in fibroblasts.

Interaction of GTPase activating proteins (GAPs) with p21ras measured by a novel fluorescence anisotropy method. Essential role of Arg-903 of GAP in activation of GTP hydrolysis on p21ras.

Ras GTPase activating proteins (GAPs) contain an invariant motif, -FLR-, within the most conserved region of their catalytic domains. Certain mutations in this motif have greatly reduced activity (Skinner, R. H., Bradley, S., Brown, A. L., Johnson, N. J., Rhodes, S., Stammers, D. K., and Lowe, P. N. (1991) J. Biol. Chem. 266, 14163-14166), but it was not determined whether the reduced activity was due to loss of binding or impaired catalysis. In order to address this question, we have developed a simple physical method to study formation of GAP.p21ras complexes. This utilizes the increase of fluorescence anisotropy upon binding of GAP to p21ras complexed with 2'(3')-O-(N-methylanthraniloyl) (mant) derivatives of guanine nucleotides. Dissociation constants obtained for the catalytic domains of either p120-GAP (GAP-344) or neurofibromin (NF1-GRD) with normal and Leu-61 p21ras proteins are comparable with those obtained by kinetic methods. In the course of these studies, we found, in contrast to previous observations, that both GAP and NF1-GRD can weakly activate the GTPase of Leu-61 mutant p21, showing that Gln-61 is not absolutely required for the stimulation of GTPase activity by GAPs. The fluorescence anisotropy method allowed us to show that mutation of Arg-903, within the FLR motif of GAP, can result in protein defective in catalysis but not in binding to p21ras. These data suggest a direct role for this residue in catalyzing GTP hydrolysis on p21ras, possibly by contributing a catalytic group to the p21 active site. This method is independent of the catalytic activity of the proteins, and so it could be extended generally to the measurement of binding of effector molecules, exchange factors, or other macromolecules to guanine nucleotide-binding proteins.

Use of the Glu-Glu-Phe C-terminal epitope for rapid purification of the catalytic domain of normal and mutant ras GTPase-activating proteins.

The C-terminal catalytic domain (residues 704-1047) of the human ras GTPase-activating protein (GAP) has been engineered so as to incorporate the tripeptide, Glu-Glu-Phe, at its C terminus. This motif is recognized by the commercially available YL1/2 monoclonal antibody to alpha-tubulin and has previously been used for the immunoaffinity purification of HIV enzymes engineered to contain this epitope (Stammers, D. K., Tisdale, M., Court, S., Parmar, V., Bradley, C., and Ross, C. K. (1991) FEBS Lett. 283, 298-302). The engineered GAP catalytic domain (GAP-344) was obtained in high yield and purity from Escherichia coli extracts by means of a single affinity column of immobilized YL1/2, eluted under mild conditions with the dipeptide, Asp-Phe. The protein had similar activity to that previously described for full-length GAP, suggesting that the addition of the epitope did not grossly affect the activity. R903K and L902I mutants of GAP-344 were constructed, and the immunoaffinity purification procedure allowed their rapid characterization. The R903K mutant had less than 3% the activity of the normal protein, whereas the L902I substitution had less than 0.5% of normal activity, suggesting an important role for Leu-902 and Arg-903, residues absolutely conserved among GAP-related proteins. This work exemplifies the general utility of the C-terminal Glu-Glu-Phe motif for the rapid purification of proteins whose function is not altered by C-terminal modification.

Characterization of recombinant human Kirsten-ras (4B) p21 produced at high levels in Escherichia coli and insect baculovirus expression systems.

Kirsten-ras is the oncogene most frequently activated in human tumors. Studies of its biological function have been limited by the nonavailability of significant amounts of the major protein product, Kirsten-ras (4B) p21. When expressed in Escherichia coli K12, the recombinant protein was rapidly cleaved upon cell lysis in the lysine-rich C terminus region, probably by the ompT protease. However, soluble full-length protein was obtained when the Kirsten-ras gene was expressed in an E. coli strain lacking the ompT gene, and also in a baculovirus/insect cell expression system. Additionally, the baculovirus/insect cell system produced about half of the Kirsten-ras protein in a membrane-associated form, which was post-translationally modified by polyisoprenylation and carboxyl-methylation. A C-terminally truncated form (residues 1-166) was also expressed at high levels in E. coli for x-ray crystallographic studies. The kinetics of GDP release and of GTP hydrolysis of the purified proteins are similar to those of the corresponding Harvey-ras proteins, though there are small differences in the relative affinities for GDP and GTP. Biological activity of full-length Kirsten Val-12 p21 was demonstrated by microinjection into Swiss 3T3 cells, resulting in morphological transformation, with a lower potency than that of Harvey Val-12 protein.

Expression and characterization of the Ha-ras p21 protein produced at high levels in the insect/baculovirus system.

Normal and mutated cDNAs of Ha-ras have each been cloned into a standard (pAc373) and a novel (p36C) baculovirus transfer vector and introduced via homologous recombination into the genome of Autographa californica nuclear polyhedrosis virus immediately downstream of the polyhedrin promoter. Spodoptera frugiperda cells infected with recombinant virus containing the normal Ha-ras gene express very high levels of ras p21 protein (approximately 20% of total cell protein), whereas the mutant protein was expressed at considerably lower levels. Molecular analysis showed that this was most likely due to a post-transcriptional event. The expression vector p36C produced considerably higher levels of recombinant p21 compared to the more commonly used pAc373. The majority of the normal ras p21 protein is soluble, cytoplasmic, and appears to be nonacylated. However, about 10% of the p21 associates with the membrane fraction of infected cells and migrates as a slightly faster band on gels. Furthermore, this band is sensitive to hydroxylamine treatment and shows specific incorporation of [3H]palmitate, strongly suggesting that it is the palmitoylated form of p21, which is the biologically active form of the protein. Both the soluble and membrane-associated p21 have been purified to homogeneity under nondenaturing conditions, the latter in the presence of detergents. The isolation of native palmitoylated p21 has not been reported previously. The difference in hydrophobicity between these two proteins has been demonstrated by Triton X-114 partitioning. The use of the insect/baculovirus expression system to express relatively high levels (20 mg/liter) of palmitoylated p21 should aid experiments to resolve the structural and functional properties of this molecule.

Mutations within the 23S rRNA coding sequence of E. coli which block ribosome assembly.

We have used site specific mutagenesis in vitro to construct a set of deletion mutations within the 5' region of a cloned 23S rRNA gene. In contrast to previously studied mutations in this gene, some of these deletions prevent the incorporation of 23S rRNA into ribosomal particles. This result is discussed in terms of a model in which interaction with the assembly initiator protein, L24, is perturbed.

Biochemical characterization of resistance determinants cloned from antibiotic-producing streptomycetes.

Determinants of antibiotic resistance have been cloned from four antibiotic-producing streptomycetes into Streptomyces lividans. Biochemical analyses of resistant clones revealed the presence of enzymes that had previously been characterized as likely resistance determinants in the producing organisms. These included: 23S rRNA methylases from S. azureus and S. erythreus, which confer resistance to thiostrepton and erythromycin, respectively; viomycin phosphotransferase from S. vinaceus; and aminoglycoside phosphotransferase and acetyltransferase from the neomycin producer S. fradiae. In general, the levels of antibiotic resistance of the clones were similar to those of the producing organisms. Although the two aminoglycoside-modifying enzymes from S. fradiae could independently confer only low-level resistance to neomycin, the presence of both enzymes in the same strain resulted in a level of resistance comparable with that of the producing organism.

Resistance to the antibiotics viomycin and capreomycin in the Streptomyces species which produce them.

Viomycin capreomycin, antibiotics produced by Streptomyces vinaceus and S. capreolus respectively, are potent inhibitors of bacterial protein synthesis. Although these organisms are highly tolerant of their own products in vivo, their ribosomes are fully sensitive to the action of the drugs in vitro. However, they processes novel, antibiotic-inactivating enzymes (viomycin phosphotransferase, capreomycin phosphotransferase, capreomycin acetyltransferase) which, in addition to possible biosynthetic roles, may contribute to the resistances observed in vivo.