Alternative selectable markers for potato transformation using minimal T-DNA vectors

Alternative selection systems for plant transformation are especially valuable in clonal crops, such as potato (Solanum tuberosum L.), to pyramid transgenes into the same cultivar by successive transformation events. We have modified the pGPTV series of binary vectors to construct pMOA1 to pMOA5, resulting in a series of essentially identical binary vectors except for the presence of different selectable marker genes. These selectable marker genes are tightly inserted between the left and right T-DNA borders and confer resistance to kanamycin (nptII), hygromycin (hpt), methotrexate (dhfr), phosphinothricin (bar), or phleomycin (ble). The T-DNA of all the vectors is based on the minimal features necessary for plant transformation, with no extraneous DNA segments that may be unacceptable to regulatory authorities for general release of transgenic plants. A series of unique restriction sites exists between the right border and each selectable marker gene for subsequent insertion of useful genes. We have also developed improved culture procedures for potato transformation and used the pMOA1 to pMOA5 binary vectors to define stringent selection conditions for each marker gene. Combining these advances improved the frequency of recovering transformed potato plants while maintaining a low frequency of escapes. The relative efficiency of recovering transgenic potato lines with each selectable marker gene can be summarised as: kanamycin resistance>hygromycin resistance>phosphinothricin resistance>phleomycin resistance>methotrexate resistance.     

Duration of the hemodynamic effects of N(G)-nitro-L-arginine methyl ester in vivo.

The objective of this study was to quantify the duration of the hemodynamic activity of N(G)-nitro-l-arginine methyl ester (l-NAME) in a variety of different tissues following a single bolus injection of this nitric oxide synthase inhibitor to healthy rats. l-NAME (15 micromol x kg(-1)) was injected (ip) into rats to produce maximal inhibition of endothelial cell NOS. Animals were subsequently anesthetized and blood flow was quantified using the radioactive microsphere/reference organ technique. At 1 h following a single bolus injection of l-NAME blood flow was reduced to the entire gastrointestinal tract, pancreas, and liver. Three hours following l-NAME administration, blood flow to the stomach and upper small intestine had returned to pretreatment levels; however, blood flow to the jejunum, ileal-jejunal junction, and colon remained significantly reduced. Splenic blood flow was significantly reduced and hepatic arterial blood flow was further reduced at this time as well. After 6 h following l-NAME administration, blood flow in all organs had completely recovered to control levels. Although cardiac index and total peripheral resistance had also returned to preinjection values at this time, mean arterial pressure remained elevated at 6 h posttreatment. Blood flow to the brain, lungs, and psoas muscle were unaffected by l-NAME administration at any time point. Taken together, these data demonstrate a differential regulation of vascular tone by NO in different vascular beds and, depending upon the organ system in question, the vasoactive activity of l-NAME may last from 3 to 6 h following a single bolus injection of this NOS inhibitor.     

An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.     

Has vertebrate chemesthesis been a selective agent in the evolution of arthropod chemical defenses?

Arthropods use a variety of chemical substances to repel potential predators, but how did they arrive at the suite of chemicals that they use? One way to explore this question is to map chemically defended arthropod species in a multidimensional "compound" space. Clustering within this space indicates species that share similar combinations of chemical compounds and can reflect a phylogenetic signal, common biochemical pathways, or both. More important for this study, clustering can help to identify allomone targets. We herein compare common arthropod allomones with known vertebrate trigeminal irritants. We argue that the degree of overlap between these two groups of compounds indicates that chemesthesis was an important determining factor in the evolution of many arthropod allomones. The multidimensional scaling methods used may also allow the identification of new irritant receptors.     

Maize Brittle Stalk2-Like3, encoding a COBRA protein,functions in cell wall formation and carbohydrate partitioning

Carbohydrate partitioning from leaves to sink tissues is essential for plant growth and development. The maize (Zea mays) recessive carbohydrate partitioning defective28 (cpd28) and cpd47 mutants exhibit leaf chlorosis and accumulation of starch and soluble sugars. Transport studies with ¹⁴C-sucrose (Suc) found drastically decreased export from mature leaves in cpd28 and cpd47 mutants relative to wild-type siblings. Consistent with decreased Suc export, cpd28 mutants exhibited decreased phloem pressure in mature leaves, and altered phloem cell wall ultrastructure in immature and mature leaves. We identified the causative mutations in the Brittle Stalk2-Like3 (Bk2L3) gene, a member of the COBRA family, which is involved in cell wall development across angiosperms. None of the previously characterized COBRA genes are reported to affect carbohydrate export. Consistent with other characterized COBRA members, the BK2L3 protein localized to the plasma membrane, and the mutants condition a dwarf phenotype in dark-grown shoots and primary roots, as well as the loss of anisotropic cell elongation in the root elongation zone. Likewise, both mutants exhibit a significant cellulose deficiency in mature leaves. Therefore, Bk2L3 functions in tissue growth and cell wall development, and this work elucidates a unique connection between cellulose deposition in the phloem and whole-plant carbohydrate partitioning.

Mutations in Bk2L3 result in dwarfed plants with decreased anisotropic cell growth, cellulose deposition, phloem pressure, sucrose export, and carbohydrate hyperaccumulation in mature maize leaves.     

Sensitivity of heat-stressed yeasts to essential oils of plants.

Eight strains of yeasts (Candida lipolytica, Debaryomyces hansenii, Hansenula anomala, Kloeckera apiculata, Lodderomyces elongisporus, Rhodotorula rubra, Saccharomyces cerevisiae, and Torulopsis glabrata) were examined for changes in sensitivity to eight essential oils of plants (allspice, cinnamon, clove, garlic, onion, oregano, savory, and thyme) after being sublethally heat stressed. With the exception of garlic oil for all test yeasts, onion oil for S. cerevisiae, and oregano oil for R. rubra, the essential oils at concentrations of up to 200 ppm in recovery media did not interfere with colony formation by unheated cells. However, some oils, at concentrations as low as 25 ppm in recovery media, reduced populations of sublethally heat-stressed cells compared to populations recovered in media containing no test oils. This demonstrates that the yeasts were either metabolically or structurally damaged as a result of being exposed to elevated temperatures and that essential oils prohibited repair of injury. The size (diameter) of colonies produced on oil-supplemented recovery agar by heat-stressed cells was reduced compared to that observed on unsupplemented agar. Pigment production by heated R. rubra was inhibited by oils of oregano, savory, and thyme, but enhanced by garlic and onion oils. The influence of essential oils on survival of yeasts in thermally processed foods and in the enumeration of stressed cells in these foods should not be minimized.