In-Plant Protection against by Production of Long hpRNA in Chloroplasts.

Expressing double-stranded RNA (dsRNA) in transgenic plants to silence essential genes within herbivorous pests is referred to as -kingdom RNA interference (TK-RNAi) and has emerged as a promising strategy for crop protection. However, the dicing of dsRNA into siRNAs by the plant's intrinsic RNAi machinery may reduce this pesticidal activity. Therefore, genetic constructs, encoding ∼200 nt duplex-stemmed-hairpin (hp) RNAs, targeting the acetylcholinesterase gene of the cotton bollworm, , were integrated into either the nuclear or the chloroplast genome of Undiced, full-length hpRNAs accumulated in transplastomic lines of and conferred strong protection against herbivory while the hpRNAs of nuclear-transformed plants were processed into siRNAs and gave more modest anti-feeding activity.

Preliminary safety assessment of a membrane-bound delta 9 desaturase candidate protein for transgenic oilseed crops.

A gene encoding delta 9 desaturase (D9DS), an integral membrane protein, is being considered for incorporation into oilseed crops to reduce saturated fatty acids and thus improve human nutritional value. Typically, a safety assessment for transgenic crops involves purifying heterologously produced transgenic proteins in an active form for use in safety studies. Membrane-bound proteins have been very difficult to isolate in an active form due to their inherent physicochemical properties.

Photorhabdus luminescens toxins ADP-ribosylate actin and RhoA to force actin clustering.

The bacterium Photorhabdus luminescens is mutualistically associated with entomopathogenetic nematodes. These nematodes invade insect larvae and release the bacteria from their intestine, which kills the insects through the action of toxin complexes. We elucidated the mode of action of two of these insecticidal toxins from P.

A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression.

Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6.

Characterization of plant beta-ureidopropionase and functional overexpression in Escherichia coli.

Pyrimidine bases are rapidly catabolized in growing plant tissues. The final enzyme of the catabolic pathway, beta-ureidopropionase (beta-UP; EC 3.5.

Isoxaben Inhibits the Synthesis of Acid Insoluble Cell Wall Materials In Arabidopsis thaliana.

The effect of the herbicide isoxaben on the incorporation of radiolabeled glucose, leucine, uracil, and acetate into acid insoluble cell wall material, protein, nucleic acids, and fatty acids, respectively, was measured. Dichlobenil, cycloheximide, actinomycin D, and cerulenin, inhibitors of the incorporation of these precursors into these macromolecular components, functioned as expected, providing positive controls. The incorporation of radiolabeled glucose into an acid insoluble cell wall fraction was severely inhibited by isoxaben at nanomolar concentrations.

A Second Locus, Ixr B1 in Arabidopsis thaliana, that Confers Resistance to the Herbicide Isoxaben.

An isoxaben resistant mutant of Arabidopsis thaliana is described whose locus, Ixr B1, is unlinked genetically to the previously described resistance locus Ixr A (DR Heim, JL Roberts, PD Pike, IM Larrinua [1989] Plant Physiol 90: 146-150). A cross of strains each homozygous for one of these two resistance loci gives rise to some isoxaben sensitive F(2) progeny. Growth curves versus isoxaben of this mutant, its F(1) progeny and the wild-type parent strain showed that this locus displays a weakly codominant Mendelian phenotype.

Primary Site of Action of Amitrole in Arabidopsis thaliana Involves Inhibition of Root Elongation but Not of Histidine or Pigment Biosynthesis.

Interference with histidine metabolism, inhibition of pigment biosynthesis, or both have been the principal candidates for the primary site of action of 3-amino 1,2,4-triazole (amitrole). Arabidopsis thaliana is sensitive to 1,2,4-triazole-3-alanine, a feedback inhibitor of histidine biosynthesis, and this effect is reversed by histidine. The combination of triazolealanine and histidine, however, does not reverse the herbicidal effect of amitrole.

Mutation of a Locus of Arabidopsis thaliana Confers Resistance to the Herbicide Isoxaben.

Mutants resistant to the herbicide N-(3-[1-ethyl-1-methylpropyl]-5-isoxazolyl)-2,6,dimethoxybenzamide (isoxaben) were recovered from an M2 population of Arabidopsis thaliana. Two of these mutants, DH47 and DH48, had a high level of resistance in the homozygous state. Crosses of these mutants to marker strains, and to each other, showed that each contained a mutation at a single locus tightly linked to lutescens, a marker on the fifth chromosome of A.

Nucleotide sequence, promoter analysis, and linkage mapping of the unusually organized operon encoding ribosomal proteins S7 and S12 in maize chloroplast.

The nucleotide sequence of the operon encoding maize chloroplast ribosomal protein genes S7 and S12 and the promoter activity of a chimeric construct of the -10/-35 sequence of this operon (attached to a promoterless chloramphenicol acetyltransferase gene) have been determined. This operon occurs in the chloroplast genome divided in two parts: part A contains exon 1 of rpS12 (encoding the N-terminal 38 amino acid residues), whereas part B has the following structure: promoter-rpS12 (exon 2 + intron + exon 3)-spacer-rpS7-terminator. Part A is located at the approximate coordinate position 41000, whereas two copies of part B are located at two distant locations in the genome at coordinate positions 18700 and 120200.

A detailed restriction endonuclease site map of theZea mays plastid genome.

Fragments produced by partial digestion of plastid DNA fromZea mays withEco RI were cloned in Charon 4A. A circular, fine structure physical map of the plastid DNA was then constructed from restriction endonucleaseSal I,Pst I,Eco RI, andBam HI recognition site maps of cloned overlapping segments of the plastid genome. These fragments were assigned molecular weights by reference to size markers from both pBR322 and lambda phage DNA.

The maize chloroplast genes for the beta and epsilon subunits of the photosynthetic coupling factor CF1 are fused.

We have cloned and sequenced the maize chloroplast genome fragment Eco RI e which contains the 2.2 kb transcript previously reported (Link, G. and Bogorad, L.

Accessibility of guanine at position 44 in the invariant sequence 5'CCG44AAC3' of Escherichia coli 5S RNA to reaction with kethoxal.

The reaction of Escherichia coli ribosomes with beta-ethoxy-alpha-ketobutyraldehyde (kethoxal) in a buffer containing 50--100 mM Tris.HCl at pH 7.4, 50 mM NH4Cl, and 5 mM Mg(OAc)2 readily released the 5S RNA from the ribosomes.

Action of Nalidixic Acid on Chloroplast Replication in Euglena gracilis.

The role of light in nalidixic acid bleaching of Euglena gracilis var. bacillaris was investigated. The kinetics of loss of the chloroplast-associated DNA and the sensitivity of chloroplast replication to ultraviolet light was followed during treatment with nalidixic acid.