Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phosphate synthase: sequence analysis and manipulation to obtain glyphosate-tolerant plants

Summary 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs), the target of the herbicide glyphosate, catalyzes an essential step in the shikimate pathway common to aromatic amino acid biosynthesis. We have cloned an EPSP synthase gene from Arabidopsis thaliana by hybridization with a petunia cDNA probe. The Arabidopsis gene is highly homologous to the petunia gene within the mature enzyme but is only 23% homologous in the chloroplast transit peptide portion. The Arabidopsis gene contains seven introns in exactly the same positions as those in the petunia gene. The introns are, however, significantly smaller in the Arabidopsis gene. This reduction accounts for the significantly smaller size of the gene as compared to the petunia gene. We have fused the gene to the cauliflower mosaic virus 35 S promoter and reintroduced the chimeric gene into Arabidopsis. The resultant overproduction of EPSPs leads to glyphosate tolerance in transformed callus and plants.     

Surface areas,lengths and volumes of Picea abies (L.) Karst. needles: determination,biological variability and effect of environmental factors

Summary A method for the rapid determination of the lengths and surface areas of very large samples of needles of Picea abies (L.) Karst. using a computer-aided image analysis system was developed. Two independent methods for measuring non-destructively the volumes of individual needles and of all needles attached to a twig were devised. The surface areas and lengths of about 38000 needles sampled from the three youngest needle age-classes (1986, 1985, 1984) of 48 trees approximately 130 years old at four sites in the Fichtelgebirge mountains (N. E. Bavaria, FRG) were measured. The frequency distributions of lengths and areas for each site and age-class are given. Variability of needle size was fairly large. Even though the sites differed in climate, soil, and air pollution levels no consistent effect of these factors on needle size could be detected. Needle lengths and surface areas did not correlate with either the total chlorophyll content of the needles or the degree of crown thinning. The needle surface area (in mm²) of fully developed P. abies needles can be estimated by the empirical equation surface area = 4.440 x needle length -24.8 (r = 0.937), and the needle volume (in mm³) by needle volume = 0.208 x projected needle area ^1.353 (r = 0.969).     

Insulin-stimulated tyrosine phosphorylation of a 43 kDa protein in rat liver membranes

The insulin receptor (IR) tyrosine kinase is essential for the regulation of different cellular functions by insulin. This may occur by a direct phosphorylation of membrane and/or cytoplasmic proteins by the IR tyrosine kinase. Hence it is important to identify putative physiological substrates for the IR tyrosine kinase. In this study we found that the glycoprotein fraction from rat liver membranes contain a 43 kDa protein (pp43) which, like the -subunit of IR, is phosphorylated in an insulin-dependent manner. A 25-fold enhancement of ³²P incorporation into pp43 by insulin was found under optimal conditions. Half-maximal phosphorylation of pp43 and the -subunit of IR were attained at 66 nM and 60 nM insulin, respectively. Mn²⁺ (K_a = 1.0 mM) was much better than Mg²⁺ (K_a = 6.3 mM) in supporting pp43 phosphorylation. Insulin-stimulated phosphorylation of pp43 (t_1/2 = 3.6 min) proceeded at a much slower rate compared to that of the -subunit of IR (t_1/2 = 1.2 min). Phosphoamino acid analysis of pp43 revealed that both tyrosine and serine are phosphorylated in the ratio 4 : 1. Tyrosine, but not serine, phosphorylation was increased 12-fold by insulin. Phosphorylation of pp43 occurred on 4 major tryptic peptides. Comparison to the tryptic phosphopeptides from IR -subunit suggest that pp43 was not derived from IR -subunit by proteolysis. Our results suggest that pp43 may be an endogenous substrate for the IR tyrosine kinase.     

The GTP-binding regulatory proteins of neuroblastoma x glioma, NG108-15, and glioma, C6, cells. Immunochemical evidence of a pertussis toxin substrate that is neither Ni nor No

Amounts of the guanine nucleotide binding regulatory proteins which are also pertussis toxin substrates (such as Ni and No) were measured in rat glioma, C6BU-1, cells and in neuroblastoma X glioma, NG108-15, hybrid cells. Measurements were performed both by quantitating pertussis toxin catalyzed ADP-ribosylation and by quantitative immunoblotting with affinity purified antibodies specific for Ni or No. The amounts of pertussis toxin substrate in C6 and NG108-15 cells are 7.5 and 0.6 pmol/mg membrane protein, respectively. These levels are minimum values and higher estimates of the total amounts of N proteins in the two cells are obtained by quantitative immunoblot analysis of the beta-subunit common to all N proteins. Immunoblots with specific antibodies show that NG108-15 cells contain 3.8 pmol/mg of No and detectable but small (less than 0.1 pmol/mg) amounts of Ni. In contrast, C6 cell membranes contain no detectable No and only 0.14 pmol/mg Ni. Thus, C6 cells contain large amounts of a pertussis toxin substrate which is neither Ni nor No.     

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS : III. The Purification of Erythroid Cells by pH-Induced Density Changes

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen.