Developing a transposon tagging system to isolate rust-resistance genes from flax

Summary A line of flax, homozygous for four genes controlling resistance to flax rust, was transformed with T-DNA vectors carrying the maize transposable elements Ac and Ds to assess whether transposition frequency would be high enough to allow transposon tagging of the resistance genes. Transposition was much less frequent in flax than in Solanaceous hosts such as tobacco, tomato and potato. Transposition frequency in callus tissue, but not in plants, was increased by modifications to the transposase gene of Ac. Transactivation of the excision of a Ds element was achieved by expressing a cDNA copy of the Ac transposase gene from the Agrobacterium T-DNA 2 promoter. Progeny of three plants transformed with Ac and 15 plants transformed with Ds and the transposase gene, were examined for transposition occurring in the absence of selection. Transposition was observed in the descendants of only one plant which contained at least nine copies of Ac. Newly transposed Ac elements were observed in 25–30% of the progeny of some members of this family and one active Ac element was located 28.8 (SE=6.3) map units from the L ⁶ rust-resistance gene. This family will be potentially useful in our resistance gene tagging program.     

Epinephrine potentiates activation of human platelets by low density lipoproteins

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.     

Activation of a latent RNAase from yeast by nucleoside triphosphates

A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography. The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose. When separated from the inhibitor, the RNAase hydrolyses RNA to 5′-mononucleotides. Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP. The RNAase is inhibited by the thermolabile component only in the presence of divalent cations. The activity is recovered upon addition of 0.01 mM ATP to the mixture. The K_m for ATP is 10 μM. ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP. These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.     

Specific protection of nucleotides in the lac operator from DMS methylation and DNase I nicking by crude bacterial cell extracts

Crude bacterial cell extracts prepared from an Escherichia coli lacIq strain were shown to protect specific nucleotides in the lac operator from methylation by dimethyl sulfate (DMS) or digestion by DNase I, whereas no protection was observed using extracts prepared from a nearly isogenic lacI- strain. These experiments show that it is not necessary to use purified regulatory proteins in experiments designed to localize sequences on DNA which interact with proteins. Therefore, crude cell extracts should be useful in DNA "footprinting" experiments to define regions of DNA which bind to unknown regulatory proteins.     

Mg2+ restores membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate movements

Cellular ornithine biosynthesis could be expected to play a significant role in putrescine formation and hence in growth. Two enzymes are involved in ornithine biosynthesis: arginase and transamidinase. These enzyme activities were studied in two human melanoma cell lines differing in their K_m of diamine oxidase for putrescine and in their tumorigenicity in nude mice. Arginase activity accounts for the majority of ornithine formed in the highly tumorigenic cell line, while the majority of ornithine is derived from transamidinase action in the poorly tumorigenic cell line, with concomitant formation of methyl guanidine, a potent inhibitor of diamine oxidase.     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.     

Tyrosine Hydroxylase Regulation: Apparent Kinetic Alterations Following Incubation of Brain Slices in a Sodium-free Medium

In an attempt to determine if alterations in intraneuronal Ca2+ may regulate tyrosine hydroxylase activity, brain slices were subjected to experimental manipulations known to increase the intraneuronal concentration of free Ca2+ ions. Incubation of either striatal or olfactory tubercle slices in a Na+-free medium for 15 min at 37 degrees resulted in a marked increase in the activity of tyrosine hydroxylase present in the 20,000 g supernatant fraction of homogenates prepared from the slices. Tyrosine hydroxylase isolated from slices previously incubated in a Na+-free, choline-enriched medium or in a Na+-free, sucrose-enriched medium exhibited maximal activities when assayed at pH 6.0 and 7.0, respectively. However, the percentage stimulation of enzyme activity induced by incubation of the slices in a Na+-free medium was maximal when the enzyme assays were performed at pH 7.0. The observed increase in enzyme activity seems to be mediated by a decrease in the apparent Km of the enzyme for pteridine cofactor, regardless of whether the kinetic enzyme analyses were conducted at pH 6.0 or 7.0, and by an increase in the Ki of the enzyme for end-product inhibitor dopamine. The apparent kinetic changes in the enzyme do not seem to result from alterations in the endogenous dopamine content of the slices, and they are independent of any increase in dopamine release that might have occurred as a response to the augmented intraneuronal Ca2+ concentration. Furthermore, the activation of tyrosine hydroxylase produced by incubating slices in a Na+-free medium is observed even in slices depleted of dopamine by pretreatment of rats with reserpine 90 min before preparation of brain slices. The activation of tyrosine hydroxylase observed under these experimental conditions does not seem to be mediated by cAMP or by a cAMP-dependent phosphorylation process. It is suggested that the changes in tyrosine hydroxylase reported are mediated primarily by a rise in the free Ca2+ concentration within the nerve tissue. These observations are consistent with the hypothesis that the kinetic activation of tyrosine hydroxylase produced after depolarization of central dopaminergic neurons may occur through a Ca2+-dependent even other than transmitter release.     

Leishmania tropica: pathogenicity and in vitro macrophage function in strains of inbred mice.

Of seven strains of inbred mice and one hybrid that were infected intracutaneously with 5, 10, or 20 × 10⁶ active promastigotes of Leishmania tropica major, two strains (CBA/Ca and C₃H/He) recovered from the infection and their lesions healed within 3 to 5 months. The other strains, with the possible exception of C57B1/6 animals, remained infected, carrying large cutaneous ulcers throughout their lives. These included DBA/2, A/Jax, Balb/c, athymic nude mice of Balb/c origin (nu/nu) and the heterozygote Balb/c (nu⁺). The responses of C57B1/6 animals were of intermediate type with a tendency toward nonhealing at higher doses of the parasite. The cutaneous infection of athymic nude mice invariably gave rise to fulminating visceral infections and death. This condition was never observed in the other strains tested. Concanavalin A (Con A)-stimulated syngeneic or allogeneic lymphocytes of intact mice activated peritoneal macrophages of both healer and nonhealer mice, resulting in complete destruction of phagocytosed L. enriettii within 24 to 48 hr. The destruction of ingested L. tropica was confined to macrophages of healer mice and required 72 to 96 hr to reach completion. However, removal of Con A-stimulated lymphocytes from macrophage cultures and regular pulsing of the cells with a lymphokine-rich supernatant produced a state of sustained activation, resulting in destruction of L. tropica inside macrophages of both healer and nonhealer mice. The ability of Con A-stimulated lymphocytes of nonhealer animals to induce effective levels of activation in healer macrophages on one hand, and eventual destruction of L. tropica in macrophages of nonhealer mice under condition of sustained activation on the other, had indicated that so far as the in vitro situation is concerned, there is no inherent defect in lymphocytes or macrophages of nonhealer animals, although the threshold of activation necessary for killing of the parasite seems to be higher for cells of nonhealer origin.     

Schistosoma mansoni: thiol proteinase properties of adult worm "hemoglobinase".

This report presents evidence that the “hemoglobinase” from adult Schistosoma mansoni, first described by Timms and Bueding and later by Senft and his collaborators, belongs to the class of thiol proteinases. Proteolytic activity is stimulated by SH-containing compounds and inhibited by N-ethylmaleimide as well as other inhibitors of thiol proteinases. The enzyme can be partially purified by affinity chromatography using a Sepharose-linked organomercurial ligand. In addition to its activity on globin and hemoglobin, the enzyme can also be assayed with Azocoll, a general protease substrate, and by the activation of inactive trypsinogen to active trypsin. Extraction of the enzyme is enhanced by the addition of the nonionic detergent Triton X-100.