The early events in legume nodulation by Rhizobium spp. involve a conserved gene cluster known as the common nod region. A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium japonicum nodDABC-acZ translational fusion was constructed and used to monitor nod gene expression in response to soybean root extract. Two inducing compounds were isolated and identified. Analysis using ultraviolet absorption spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 4',7-dihydroxyisoflavone (daidzein) and 4',5,7-trihydroxyisoflavone (genistein). Induction was also seen with some, but not all, of the flavonoid compounds that induce nod genes in fast-growing Rhizobium strains that nodulate clover, alfalfa, or peas. When pEA2-21 was introduced into Rhizobium trifohii, it was inducible by flavones but not by daidzein and genistein. In Rhizobium fredii, pEA2-21 was induced by isoflavones and flavones. Thus, the specificity of induction appears to be influenced by the host-strain genome.Members of the bacterial genus Rhizobium form symbiotic associations with leguminous plants that result in the formation of nitrogen-fixing root nodules. In three agronomically important Rhizobium/legume associations, R. trifolii/clover, R. meliloti/alfalfa, and R. leguminosarum/pea, important bacterial nodulation genes (1-4) and plant compounds that induce them (5-7) have been identified. In these associations flavones (5-7) or flavanones (7) have been found to induce the nodABC genes, as well as other nod genes involved in host specificity (1). Isoflavones have been found to inhibit the induction of nodABC in R. leguminosarum (7).The Bradyrhizobium japonicum/soybean symbiosis is of considerable agricultural importance. In contrast to Rhizobium spp., Bradyrhizobium species are slow-growing (8), nitrogen-fixation and nodulation genes are located on the chromosome (9) and not on plasmids (10)(11)(12), and less is known about the genetic requirements for nodulation (13)(14)(15). In particular, the compounds produced by the soybean host that interact with the common nod genes have not been characterized.In this study, a nodABC-lacZ translational fusion was used to monitor nod gene expression in B. japonicum in response to soybean root extract. Two major components from soybeans (Glycine max cv. Williams) were isolated that induced the expression of the nodABC-lacZ fusion when it was present in the soybean-nodulating bacteria B. japonicum and Rhizobium fredii, but not when it was present in R. trifolii. MATERIALS AND METHODSStrains and Plasmids. Standard procedures (16) were used for DNA manipulations. A HindIII fragment containing the nod region of B. japonicum USDA 123 was cloned in both orientations into the single HindIII site of plC19R (17). BamHI-Bgl II fragments containing the nod genes and flanking polylinker sequences were then cloned into the BamHI site of the broad-host-range vector pGD926 (18) resulting in pEA2-21 and pEA4-10 (Fig. 1) (19) agar and germinated for 3 days in the d...

The insecticidal crystal protein gene of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis has been isolated, and the nucleotide sequence has been determined. A total DNA library from var. tenebrionis was made in the plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corresponding to a stretch of nine N-terminal amino acids of a tryptic fragment of purified crystal protein of var. tenebrionis as a probe, recombinant colonies were screened by in situ hybridization for the presence of the crystal protein gene. Positive clones obtained from this screening were further tested for toxicity. One recombinant, NSBP544 (which contained a 5.9-kilobase BamHI insert), was toxic to larvae of Colorado potato beetle. Immunoblot analysis revealed that this clone produces two crystal-specific antigens of 65 and 73 kDa as do sporulating var. tenebrionis cells. However, purified crystal inclusions from var. tenebrionis contain a primary peptide component of 65 kDa. A 1932-base-pair open reading frame with a coding capacity of 73,119 Da has been identified by nucleotide sequencing analysis of the cloned crystal protein.In addition, mung bean nuclease mapping indicates that transcription of the crystal protein of var. tenebrionis initiates 130 base pairs upstream from the translational start site. Southern blot analysis using an internal 0.7-kilobase EcoRI fragment of pNSBP544 as a probe revealed that the crystal protein gene is located on a 90-MDa plasmid.Bacillus thuringiensis is unique in its ability to produce proteinaceous, crystalline inclusions (8 endotoxins) during the process of sporulation, which are found to be highly toxic to larvae of several lepidopteran insect pests of agricultural importance (1). Numerous isolates of B. thuringiensis strains belonging to over two dozen distinct flagellar serotypes have been isolated and classified to date (2). The crystal proteins of several of these varieties have a rather narrow host range and hence are used commercially as very selective biological insecticides. Strains of B. thuringiensis that are toxic to larvae of dipteran insects have also been isolated recently (3,4).The 3-endotoxin genes of various B. thuringiensis varieties have been cloned by several research groups (5-10). Kronstad et al. (11) have shown that these genes are generally located on one or more large plasmids. These authors have also demonstrated that while several lepidopteran-active genes share extensive nucleotide sequence similarity, no homology exists between the lepidopteran and dipteran toxin genes.Since several agronomically important insect pests belong to the order Coleoptera (e.g., Colorado potato beetle, boll weevil, and corn root worm), an extensive search for coleopteran-toxic B. thuringiensis strains has been undertaken by several laboratories. Such efforts have resulted in the recent isolation oftwo strains ofB. thuringiensis, namely var. tenebrionis (12) Peptide Analysis and Oligonucleotide Synthesis. Crystal inclusions were purified from a crude spore/cr...

The maize 15‐Kd zein structural gene was placed under the regulation of French bean β‐phaseolin gene flanking regions. Agrobacterium tumefaciens‐mediated transformation was used to insert the chimeric phaseolin–zein gene into the tobacco genome. Transgenic plants synthesized zein in a tissue‐specific manner during the latter half of seed development. Transcription of the chimeric gene was initiated in phaseolin‐derived sequences, and was terminated within the phaseolin gene 3′ flanking region. Both zein‐ and phaseolin‐derived polyadenylation signals were used in the processing of zein RNA in transgenic plant seeds. Zein accumulation, though subject to an 80‐fold variation among 19 plants tested, could reach as much as 1.6% of the total seed protein in several plants. In developing tobacco seeds, zein was correctly processed by the removal of a 20‐amino‐acid signal peptide. Electron microscope immunogold localization of the zein expressed in embryo and endosperm tissue indicates that the monocot protein accumulates in the crystalloid component of vacuolar protein bodies. The density of gold label over the protein bodies is several fold greater in the embryo than the endosperm. Zein is found in roots, hypocotyls and cotyledons of germinating transgenic tobacco seeds.

A series of hydroxamates was prepared from an aminoproline scaffold and tested for efficacy as matrix metalloproteinase (MMP) inhibitors. Detailed SAR for the series is reported for five enzymes within the MMP family, and a number of inhibitors, such as compound 47, display broad-spectrum activity with sub-nanomolar potency for some enzymes. Modifications of the P1' portion of the molecule played a key role in affecting both potency and selectivity within the MMP family. Longer-chain aliphatic substituents in this region of the molecule tended to increase potency for MMP-3 and decrease potency for MMP-1, as exemplified by compounds 48-50, while aromatic substituents, as in compound 52, generated broad-spectrum inhibition. The data is rationalized based upon X-ray crystal data which is also presented. While the in vitro peroral absorption seemed to be less predictable, it tended to decrease with longer and more hydrophilic substituents. Finally, a rat model of osteoarthritis was used to evaluate the efficacy of these compounds, and a direct link was established between their pharmacokinetics and their in vivo efficacy.

2-Aryl-3-pyrimidinyl based tumor necrosis factor-alpha (TNF-alpha) inhibitors, which contain a novel bicyclic pyrazolone core, are described. Many showed low-nanomolar activity against lipopolysaccharide-induced TNF-alpha production in monocytic cells. Secondary screening data are presented for the pyrimidinyl bicyclic pyrazolones. Several of these analogues showed good oral bioavailability in rat and efficacy in the rat iodoacetate in vivo model.