Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression

In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

Nuclear genome characterization of the carrageenophyteAgardhiella subulata (Rhodophyta)

DNA reassociation kinetics were used to determine nuclear genome organization and complexity inAgardhiella subulata (Gigartinales, Rhodophyta). Results indicate the presence of three second-order components corresponding to fast (22%), intermediate (68%) and slow (10%) fractions. Thus, the genome consists of 90% repetitive sequences. Microspectrophotoometry with the DNA-localizing fluorochrome DAPI was used to confirm ploidy level differences in the gametophytic and tetrasporophytic phases. Results indicate that meiosis occurs during tetrasporogenesis. Comparison of mean nuclear DNA (I_f) values to chicken erythrocytes (RBC) resulted in an estimate of 0.9 pg/2C genome forAgardhiella. Karyological studies using aceto-orcein revealed a chromosome complement of 2N = 44 in carposporangia and the presence of 22 bivalents during diakinesis of tetraspore mother cells.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.     

Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport.

Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli. Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane. Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose. We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues. Residues in an area on the cleft side of the molecule including both domains have effects on transport. A portion of the area involved in transport is also essential to chemotactic function. On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically.     

Role of protein phosphorylation and inositol phospholipid turnover in rat parotid gland proliferation

The involvement of protein phosphorylation in isoproterenol (ISO)-mediated proliferation in the rat parotid gland was investigated by labeling the cells with [³²P] orthophosphate. An increased (4–6 fold) incorporation of the radiolabel was noted in the total parotid gland homogenates of ISO-treated animals when compared to controls. Plasma membrane, nuclear membrane and cytoplasm were isolated, the proteins separated by SDS/PAGE and the phosphoproteins detected by autoradiography. Two phosphoproteins with apparent M_r of 45 and 170 kDa were identified in the cytoplasm while the 170 kDa phosphoprotein also appeared as part of plasma membrane. Transfer of these proteins to nitrocellulose followed by Western blot detection with an antiphosphotyrosine monoclonal antibody showed reactivity with the 170 kDa region of the plasma membrane and cytoplasm. Separate in vitro studies involving incubations of rat parotid slices with 0.2 mM ISO and [³H] myo-inositol for 1 min induced inositol phosphate hydrolysis resulting in a significant increase in inositol-bis and -tris phosphate production. Inositol phosphate production can be blocked by pre-incubation with a mixed -adrenergic receptor antagonist but not with physiological concentrations of - or ₁-specific adrenergic receptor antagonists, indicating the ISO effects are mediated through the ₂-adrenergic receptors. The inclusion of calmodulin antagonists along with ISO prevented the expression of cell-surface galactosyltransferase and retarded gland hypertrophy and hyperplasia. These results suggest that ISO treatment leads to the phosphorylation of target proteins which may be involved in signal transduction pathways leading to cell proliferation.Abbreviations InsP₁, InsP₂, InSP₃ inositol mono-, bis-, and tris-phosphates - UDP Uridine diphosphate - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate - TFP Trifluoperazine - P-tyr phosphotyrosine - Gal Tase galactosyltransferase     

Cellular Localization of the Molecular Forms of Acetylcholinesterase in Primary Cultures of Rat Sympathetic Neurons and Analysis of the Secreted Enzyme

The secretion and cellular localization of the molecular forms of acetylcholinesterase (AChE) were studied in primary cultures of rat sympathetic neurons. When cultured under conditions favoring a noradrenergic phenotype, these neurons synthesized and secreted large quantities of the tetrameric G4, and the dodecameric A12 forms, and minor amounts of the G1 and G2 forms. When these neurons adopted the cholinergic phenotype, i.e., in the presence of muscle-conditioned medium, the development of the cellular A12 form was completely inhibited. These neurons secreted only globular, mainly G4, AChE. Both cellular and secreted A12 AChE in adrenergic cultures aggregated at an ionic strength similar to that of the culture medium, raising the hypothesis that this form was associated with a polyanionic component of basal lamina. In noradrenergic neurons, 60-80% of the catalytic sites were exposed at the cell surface. In particular, 80% of G4 form, but only 60% of the A12 form, was external, demonstrating for the A12 form a sizeable intracellular pool. The hydrophobic character of the molecular forms was studied in relation to their cellular localization. As in muscle cells, most of the G4 form was membrane-bound. Whereas 76% of the cell surface A12 form was solubilized in the aqueous phase by high salt concentrations, only 50% of the intracellular A12 form was solubilized under these conditions. The rest of intracellular A12 could be solubilized by detergents and was thus either membrane-bound or entrapped in vesicles originating from, e.g., the Golgi apparatus.     

Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

Localization of a base-paired interaction between small nuclear RNAs U4 and U6 in intact U4/U6 ribonucleoprotein particles by psoralen cross-linking

The small nuclear RNAs U4 and U6 display extensive sequence complementarity and co-exist in a single ribonucleoprotein particle. We have investigated intermolecular base-pairing between both RNAs by psoralen cross-linking, with emphasis on the native U4/U6 ribonucleoprotein complex. A mixture of small nuclear ribonucleoproteins U1 to U6 from HeLa cells, purified under non-denaturing conditions by immune affinity chromatography with antibodies specific for the trimethylguanosine cap of the small nuclear RNAs was treated with aminomethyltrioxsalen. A psoralen cross-linked U4/U6 RNA complex could be detected in denaturing polyacrylamide gels. Following digestion of the cross-linked U4/U6 RNA complex with ribonuclease T1, two-dimensional diagonal electrophoresis in denaturing polyacrylamide gels was used to isolate cross-linked fragments. These fragments were analysed by chemical sequencing methods and their positions identified within RNAs U4 and U6. Two overlapping fragments of U4 RNA, spanning positions 52 to 65, were cross-linked to one fragment of U6 RNA (positions 51 to 59). These fragments show complementarity over a contiguous stretch of eight nucleotides. From these results, we conclude that in the native U4/U6 ribonucleoprotein particle, both RNAs are base-paired via these complementary regions. The small nuclear RNAs U4 and U6 became cross-linked in the deproteinized U4/U6 RNA complex also, provided that small nuclear ribonucleoproteins were phenolized at 0 degree C. When the phenolization was performed at 65 degrees C, no cross-linking could be detected upon reincubation of the dissociated RNAs at lower temperature. These results indicate that proteins are not required to stabilize the mutual interactions between both RNAs, once they exist. They further suggest, however, that proteins may well be needed for exposing the complementary RNA regions for proper intermolecular base-pairing in the course of the assembly of the U4/U6 RNP complex from isolated RNAs. Our results are discussed also in terms of the different secondary structures that the small nuclear RNAs U4 and U6 may adopt in the U4/U6 ribonucleoprotein particle as opposed to the isolated RNAs.     

Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants

The response of tomato plants to various chilling treatments was studied using two approaches for the measurement of photosynthetic activity. One involved the use of a portable fluorometer for the measurement of in-vivo chlorophyll fluorescence, while the other employed a newly introduced photoacoustic system which allowed changes in oxygen evolution to be followed in a leaf disc. A strong correlation was found between results obtained by each system and those obtained by a conventional open gas-exchange system for the determination of CO₂ uptake. Both systems of measurements could readily distinguish between the effects of chilling in the dark (at 3° C for 18 h) and chilling at high photon flux density (2000 mol m^-2 s^-1 for 5h at 5° C). Chilling in the dark had practically no effect on the quantum yield of oxygen evolution, chlorophyll fluorescence or CO₂ uptake, while chilling at excessively high photon flux density resulted in a sharp reduction (50–70%) in the quantum yields obtained. The results support the view that photosystem II cannot be the primary site of damage by chilling in the dark, although it is significantly affected by chilling at high light intensity.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PA photoacoustic - PFD photon flux density - PSII photosystem II