Direct gene transfer to plants

Evidence for direct, gene-mediated stable genetic transformation of plant cells of Nicotiana tabacum is presented. A selectable hybrid gene comprising the protein coding region of the Tn5 aminoglycoside phosphotransferase type II gene under control of cauliflower mosaic virus gene VI expression signals was introduced into plant protoplasts as part of an Escherichia coli plasmid. The gene was stably integrated into plant genomic DNA and constitutively expressed in selected, drug resistant, protoplast-derived cell clones. The mode of integration of the foreign gene into the plant genome resembled that observed for DNA transfection of mammalian cells. Plants regenerated from transformed cell lines were phenotypically normal and fertile, and they maintained and expressed the foreign gene throughout the development of vegetative and generative organs. Microspores, grown in anther culture, developed into resistant and sensitive haploid plantlets. Genetic crossing analysis of one of the transformed plants revealed the presence of one dominant trait for kanamycin resistance segregating in a Mendelian fashion in the F₁ generation.     

Production of penicillins and cephalosporins in an immobilized enzyme reactor

Summary Four enzymes required for the biosynthesis of pencillins and cephalosporins by Streptomyces clavuligerus have been immobilized on an anion exchange resin. The capabilities of the system have been studied by circulation of reaction mixtures through the immobilized enzyme reactor. Within 30 min, all of the substrate -(l--aminoadipyl)-l-cysteinyl-d-valine is consumed and converted to a mixture of penicillins and cephalosporins. After 60 min the major antibiotic products are (iso)penicillin N and desacetylcephalosporin C. The activity of the immobilized enzyme reactor activity is stable to storage at temperatures below 4°C but activity is lost on repeated use.     

Cell lineage,cell-cell interaction,and segment formation in the ectoderm of a glossiphoniid leech embryo

Cell division patterns and cell-cell interactions in the germinal bands of the glossiphoniid leech Helobdella triserialis were studied with the aid of a cell lineage tracer dye. Each germinal band of the Helobdella embryo consists of five columns, or bandlets, of primary blast cells, designated as the mesodermal m bandlet and ectodermal n, o, p, and q bandlets. Primary blast cells of each ectodermal bandlet appear to undergo stereotyped, lineage-specific cell divisions. The metameric segmentation pattern of the leech thus appears to arise through a series of segmentally iterated, stereotyped cell divisions of serially homologous primary blast cell clones. Cell-cell interactions were studied by means of cell ablations. With one exception, blast cells underwent their stereotyped divisions without regard to the presence or absence of their normal neighbors. In the one exceptional case, o blast cells underwent divisions normally characteristic of p blast cells when their normal neighboring p bandlet was deleted. However, both o and p blast cells underwent their normal stereotyped divisions when their neighboring m, n, and q bandlets were deleted. It is proposed that the differential choice of pathway by the o and p blast cells depends upon their relative position with respect to each other and to a polarity cue external to the germinal band.     

Localization of corticotropin-releasing factor-containing neurons in the brain of the domestic fowl

Summary The corticotropin-releasing factor (CRF)-containing neurons were investigated in the brain of the domestic fowl by means of the peroxidase-antiperoxidase technique at the light-microscopic level. The detection of CRF-immunoreactivity was facilitated by silver intensification. CRF-containing perikarya were found in the paraventricular, preoptic and mammillary nuclei of the hypothalamus and in some extrahypothalamic areas (nuclei dorsomedialis and dorsolateralis thalami, nucleus accumbens septi, lobus parolfactorius, periaqueductal gray of the mesencephalon, nucleus oculomotorius ventralis). Immunoreactive nerve fibers and terminals were demonstrated in the external zone of the median eminence and the organum vasculosum of the lamina terminalis. These results indicate that an immunologically demonstrable CRF-neurosecretory system also exists in the avian central nervous system.     

Synthesis of tritiated 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid ([3H]DIDS) and its covalent reaction with sites related to anion transport in human red blood cells

Summary The potent and specific inhibitor of anion permeability, 4,4-diisothicyanostilbene-2,2-disulfonic acid (DIDS) was synthesized in tritiated form ([³H]DIDS) from tritiated 5-nitrotoluene-o-sulfonic acid. Its reactions with and effects on red blood cells were compared with those of a reduced form ([³H]H₂DIDS), previously used as a tracer for DIDS. The rate of covalent reaction of [³H]DIDS was substantially faster than that of [³H]H₂DIDS at all temperatures tested. With both agents, the rate of reaction was increased in alkaline media, although the response occurred at a lower pH with [³H]DIDS. On the other hand, the relationship of irreversible membrane binding to the degree of inhibition of sulfate fluxes was linear and virtually the same for both agents, with 100% inhibition associated with the binding of approximately 1.2×10⁶ molecules per cell. About 90% of the binding for each probe was to a particular membrane protein, known as band 3, equivalent to about 1 mole of agent per mole of protein.     

The fine structure of distal receptors on the labium of the aphid,Brevicoryne brassicae L. (Homoptera)

Summary Short peg receptors located at the distal tip of the aphid labium have the structure of mechanoreceptors. Each peg is innervated by a single sensory nerve which is anchored eccentrically to a basal cuticular tube and terminates in electron-dense material in the base of the peg. The arrangement and eccentric insertion of the eight pegs in the labial wall on one side of the stylet groove, with the eccentric insertions of their innervating neurones, provide a mirror image of the receptors on the opposite side. On the basis of a comparison of the structure of these receptors with that of tactile receptors for which electrophysiological data on sensitivity are available, it is possible to predict that the receptors detect both surface contact (pressure) and surface profile; and that the bilateral symmetry in the receptor arrangement facilitates the detection of vein contours which are preferred settling sites on the leaf. The structure of the dendritic terminal and its insertion is that of a well reinforced cytoskeleton designed to transmit tension to the cell membrane, in agreement with the concept that transduction is a membrane related phenomenon. The distal microtubules, fifty per-cent of which originate as well as terminate in the tubular body, are packed in electron-dense material which binds to the cell membrane. The membrane in turn is attached to cuticular components of the receptor. Abrupt changes in dimension of the dendritic outer segment may be designed to modulate the conduction of a membrane potential. On the other hand, lack of continuity in the microtubules makes these organelles poor candidates for the transduction of excitation from a distal site of stimulation to a proximal region.Supported by operating grants Nos. A 6063 and A 9856 from NRCC     

Site of bicarbonate effect in Hill reaction. Evidence from the use of artificial electron acceptors and donors

Using artificial electron donors and acceptors, it is shown here that the major HCO₃^? effect in the Hill reaction is after the “primary” electron acceptor (Q) of Photosystem II and before the site of action of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (at the plastoquinone pool). Chloroplasts in the presence of both 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, which blocks electron flow from the reduced primary acceptor Q^? to the plastoquinone pool, and silicomolybdate, which accepts electrons from Q^?, show no significant bicarbonate stimulation of electron flow. However, a 6–7-fold stimulation is clearly observed when oxidized diaminodurene, as an electron acceptor, and dibromothymoquinone, as an inhibitor of electron flow beyond the plastoquinone pool, are used. In the same chloroplast preparation no measurable effect of bicarbonate is observed in a Photosystem I reaction as monitored by electron flow from reduced diaminodurene to methyl viologen in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea. The insensitivity of the bicarbonate effect to uncouplers of photophosphorylation and the dependence of this effect on the presence of a weak acid anion and on external pH are also reported.     

Phylogenetic depth ofThermotoga maritima inferred from analysis of thefus gene: Amino acid sequence of elongation factor G and organization of theThermotoga str operon

Summary The gene (fus) coding for elongation factor G (EF-G) of the extremely thermophilic eubacteriumThermotoga maritima was identified and sequenced. The EF-G coding sequence (2046 bp) was found to lie in an operon-like structure between the ribosomal protein S7 gene (rpsG) and the elongation factor Tu (EF-Tu) gene (tuf). TherpsG, fus, andtuf genes follow each other immediately in that order, which corresponds to the order of the homologous genes in thestr operon ofEscherichia coli. The derived amino acid sequence of the EF-G protein (682 residues) was aligned with the homologous sequences of other eubacteria, eukaryotes (hamster), and archaebacteria (Methanococcus vannielii). Unrooted phylogenetic dendrogram, obtained both from the amino acid and the nucleotide sequence alignments, using a variety of methods, lend further support to the notion that the (present) root of the (eu)bacterial tree lies betweenThermotoga and the other bacterial lineages.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)