Evaluation of selectable markers for obtaining stable transformants in the gramineae

Cell suspension cultures of Triticum monococcum, Panicum maximum, Saccharum officinarum, Pennisetum americanum, and a double cross trispecific hybrid between Pennisetum americanum, P. purpureum, and P. squamulatum were tested for resistance to kanamycin, hygromycin, and methotrexate for use in transformation studies. All cultures showed high natural levels of resistance to kanamycin, in excess of 800 milligrams per liter, and variable levels of resistance to hygromycin. Methotrexate was a potent growth inhibitor at low concentrations with all species. Kanamycin and hygromycin were growth inhibitory only if added early (within 5 days after protoplast isolation and culture). Protoplasts of T. monococcum, P. maximum, S. officinarum, and the tri-specific hybrid were electroporated with plasmid DNA containing hygromycin (pMON410), kanamycin (pMON273), or methotrexate (pMON806) resistance genes. Resistant colonies were obtained at low frequencies (1 × 10⁻⁵ to 2 × 10⁻⁶) when selected under conditions which were growth inhibitory to protoplasts electroporated without DNA. Southern blot hybridization confirmed stable integration of plasmid DNA into T. monococcum using hygromycin vectors and P. maximum using the methotrexate vector with 1 to 10 copies integrated per haploid genome.     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

Signal transduction of gamma/delta T cell antigen receptor with a novel mitogenic anti-delta antibody

Human T lymphocytes express either alpha/beta- or gamma/delta-TCR in association with the CD3 complex. We have isolated a mAb, delta TCS1, that immunoprecipitated the gamma/delta-TCR heterodimer from cell lysates of Peer and Molt-13 leukemia cell lines. After dissociation of the gamma- and delta-chains of TCR by treatment with SDS, delta TCS1 specifically immunoprecipitated the delta-chain. This antibody bound to the surface of other gamma/delta-positive T cell lines and clones and was able to stimulate the proliferation of a minor cell population (0.9 to 4.0%) of resting human PBL. Upon binding to gamma/delta-TCR-bearing Molt-13 cells and PBL, delta TCS1 elicited a fura-2 Caa+ signal indicating that the gamma/delta-receptor is functionally similar to the alpha/beta-heterodimer. These data indicate that the delta TCS1 antibody recognizes an epitope on TCR delta-chain and its mitogenic activity should be useful in characterizing the functional properties of human gamma/delta-positive T lymphocytes.     

Correlation of polarized absorption spectroscopic and X-ray diffraction studies of crystalline cytosolic aspartate aminotransferase of pig hearts

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P2(1)2(1)2(1). The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the c/a polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5'-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 degrees. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals if forms a Schiff base with the co-enzyme in which the ring has tilted about 32 degrees from its original position and the polarization ratio of the 436 nm band drops from 1.6 in the free enzyme to about 0.38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Hydrazine stress in the diabetic: ornithine decarboxylase activity

Streptozotocin-induced diabetes of 7 weeks duration increased male Sprague-Dawley rat kidney ornithine decarboxylase activity by 4.8-fold but did not affect the liver enzyme. Hydrazine treatment of 4 hr duration stimulated equally kidney ornithine decarboxylase activities of nondiabetic and diabetic rats. Hydrazine treatment increased liver ornithine decarboxylase activity in the nondiabetic rat but did not increase it in the diabetic rat. Since hydrazine stimulates ornithine decarboxylase activity prior to polyamine and protein syntheses, we speculate that the lack of hydrazine stimulation of ornithine decarboxylase in the diabetic liver may be related in part to the unrestrained gluconeogenesis and depressed Kreb's cycle activity: the latter being required for protein synthesis.     

Neural function: metabolism and actions of inositol metabolites in mammalian brain

In the nervous system, a variety of cell types respond to external stimuli through the inositol lipid signalling pathways. The stimulus-coupled sequence of intracellular events has been investigated in a homogeneous model system, the cloned mammalian neural cell line NG115-401L. The neural peptide bradykinin stimulates a rapid production of identified inositol phosphate isomers and an intracellular Ca2+ discharge followed by a persistent plasma membrane influx. The temporal sequence suggests that Ins(1,4,5)P3 or Ins(1,3,4,5)P4 or both may coordinate these events in a neuronal cell, as has been suggested in other cell types. Thapsigargin, an irritant and tumour-promoting plant product, produces calcium transients in the absence of inositol phosphate production, and may provide a new tool for investigating the interactions between inositol phosphates and changes in cellular calcium homeostasis. In the 401L line, high levels of radiolabelled InsP5 and InsP6 have been detected, which has led to the evaluation of their possible occurrence and actions in normal brain. Both InsP5 and InsP6 are produced from a radiolabelled myo-inositol precursor in intact mature brain in a region-specific manner. This suggests that both inositol polyphosphates may be end products of regionally regulated biosynthetic pathways. When microinjected into a nucleus of the brainstem, or iontophoretically applied to the dorsal horn of the spinal cord, both InsP5 and InsP6, but not Ins(1,3,4,5)P4 isomers, appear to be potent neural stimulants. These results suggest that the inositol lipid signalling pathways may generate both intracellular and extracellular signals in brain.     

Stoichiometric network analysis

Stoichiometric network analysis is a systematic, general approach to the qualitative, nonlinear dynamics of chemical reaction mechanisms and other systems with stoichiometry. The advantage of a qualitative approach is that no rate constants are needed to determine qualitative features of the dynamics. If one is interested in stability, the approach yields inequalities among the steady-state concentrations and the rate of flow through sequences of important reactions. These parameters are often the ones most easily measured experimentally. By comparing such experiments with the inequalities derived from stoichiometric network analysis, one can often prove that certain mechanisms cannot account for oscillations or other types of observed dynamics. The approach covers far more than stability. The existence of steady states of zero concentration has an interesting mathematics and applies to chemical evolution. The folding of the manifold of steady states can be found by direct calculation and plays a role in switching enzymes on and off. The approach leads to theorems showing that some steady states are globally attracting or, possibly, that a region containing chaos or an oscillation is globally attracting. The subject of sensitivity analysis has been reformulated in this context. Algorithms that apply many of the theoretical results to chemical networks have been developed and combined into a computer program package.