Leaf developmental stage and tissue location affect the detection of β-glucuronidase in transgenic tobacco plants

Expression in Nicotiana tabaccum L. plants containing the w6q313w0053468/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase (GUS) gene under the control of the 35S (CaMV promoter) was affected by tissue type and ontogenic development of the leaves. GUS activity in ontogenetically younger leaves was 1003–1022 nmol 7-hydroxy-4-methylcoumarin (MU) formed mg^–1 (protein) min^–1 and in ontogenetically older leaves was only 140–198 nmol (MU) mg^–1 (protein) min^–1.     

Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly-w30/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca₃(PO₄)₂ as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl^–1 which was almost twice that without Ca₃(PO₄)₂ addition.     

Analysis of a dehiscence zone endo-polygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for roles in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth

The oilseed rape (Brassica napus) endo-polygalacturonase (endo-PG) RDPG1 is involved in middle lamella breakdown during silique opening. We investigated tissue-specific expression of RDPG1 in transgenic Arabidopsis thaliana. Cellular localization of endo-PG protein in Arabidopsis siliques was determined by immuno-electron microscopy. An Arabidopsis orthologue, ADPG1, was isolated and aligned with the sequence of RDPG1. The proximal 5w084712327v73615/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0"> sequences as well as introns are largely conserved. Analysis of the histological GUS-staining pattern of two RDPG1 promoter-GUS (w084712327v73615/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase) constructs in transgenic Arabidopsis revealed that the conserved proximal part of the 5w084712327v73615/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-flanking region directs expression in dehiscence zones of siliques and anthers, floral abscission zones and stylar tissues during pollen tube growth, branch points between stems and pedicel and expression associated with the apical meristem of seedlings, while the distal part of theRDPG1 5w084712327v73615/xxlarge8242.gif" alt="prime" align="BASELINE" BORDER="0">-flanking region contains elements involved in vascular-associated expression in petals, cotyledons and roots. Subsequent RT-PCR analysis, on RNA from the corresponding rape tissues, confirms the staining pattern revealed in transgenic Arabidopsis, thereby justifying the use of Arabidopsis as a reliable model system for analysis of oilseed rape regulatory sequences.     

AQR1 gene (ORF YNL065w) encodes a plasma membrane transporter of the major facilitator superfamily that confers resistance to short-chain monocarboxylic acids and quinidine in Saccharomyces cerevisiae

We report results on the functional analysis of Saccharomyces cerevisiae ORF YNL065w, predicted to code for a protein belonging to the poorly characterized major facilitator superfamily (MFS) of transporters that are involved in multidrug resistance (MDR). YNL065w is important for a moderate increase of yeast tolerance to ketoconazole and to the cationic dye crystal violet; it protects the cell against short-chain monocarboxylic acids (C(2)-C(6)), but not against highly liposoluble acids such as octanoic acid or the phenoxyacetic-acid herbicides 2,4-D and MCPA; it is also a determinant of resistance to the antiarrhytmic and antimalarial drug quinidine. The encoding ORF was, thus, denominated the AQR1 gene. Results obtained using an AQR1-lacZ fusion indicate that gene expression is very low and it is not stimulated under weak acid stress. The encoded putative transporter was localized in the plasma membrane by fluorescence microscopy observation of the overproduced Aqr1-GFP fusion protein distribution.     

Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in<Emphasis Type="Italic"> Xenopus laevis</Emphasis> oocytes

Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7, chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and the Drosophila melanogaster/chicken hybrid receptors SAD/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and ALS/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2. No agonist action of NTX (0.1–100 w7lvf6jd/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0">M) was observed on chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7, chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7) or heteromeric (w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2) nicotinic AChRs. Block by NTX of the chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">7, chicken w7lvf6jd/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">4w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and the SAD/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 and ALS/w7lvf6jd/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.     

Temporal dynamics of three culturable γ-Proteobacteria taxa in salt marsh sediments

Temporal dynamics of three marine w164j718427w3142/xxlarge947.gif" alt="gamma" align="MIDDLE" BORDER="0">-Proteobacteria taxa withculturable members were followed in sediment communities in southeastern USsaltmarshes using a whole-genome hybridization approach. Labeled DNA from threebacterial isolates was used to probe sediment community DNA from two saltmarshes on Sapelo Island, GA at multiple time points over a three-year period.The relative abundance of the three isolates (and their close relatives)accounted for up to 28% of the total sediment community DNA. Hybridizationsignals for the taxon represented by isolate SIGA28M (with a 16S rRNA sequencesimilarity of 97% to Vibrio proteolyticus) showedsignificant temporal variation in both marshes, varying from 1% to 28% over thethree-year period. The taxa represented by isolates SIGA172a and SIGA198 (16SrRNA sequence similarities of 90% and 92% to Shewanellafrigidimarina and Vibrio nigripulchritudo,respectively) accounted for less than 6% of the sediment community at any timepoint. The variation in relative abundance of these three groups did not appearto follow clear seasonal trends, and could not be readily correlated withenvironmental variables.     

A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E. coil

Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions. The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold. Thermostability of MeHNL was also enhanced, probably due to an increase in content of the w541r32nx4510416/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-strand secondary structure according to CD analysis. Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0.     

Chlorophyll reduction in the seed of Brassica napus with a glutamate 1-semialdehyde aminotransferase antisense gene*

Chlorophyll reduction in the seed of Brassica can be achieved by downregulating its synthesis. To reduce chlorophyll synthesis, we have used a cDNA clone of Brassica napus encoding glutamate 1-semialdehyde aminotransferase (GSA-AT) to make an antisense construct for gene manipulation. Antisense glutamate 1-semialdehyde aminotransferase gene (Gsa) expression, directed by a Brassica napin promoter, was targeted specifically to the embryo of the developing seed. Transformants expressing antisense Gsa showed varying degrees of inhibition resulting in a range of chlorophyll reduction in the seeds. Seed growth and development were not affected by reduction of chlorophyll. Seeds from selfed transgenic plants germinated with high efficiency and growth of seedlings was vigorous. Seedlings from T₂ transgenic lines segregated into three distinctive phenotypes: dark green, light green and yellow, indicating the dominant inheritance of Gsa antisense gene. These transgenic lines have provided useful materials for the development of a low chlorophyll seed variety of B. napus.