The yeast polyadenylate-binding protein (PAB1) gene acts as a disease lesion mimic gene when expressed in plants

We have expressed the gene (PAB1) encoding the yeast polyadenylate-binding protein (Pab1p) in tobacco. Plants that accumulate the Pab1p display a range of abnormalities, ranging from a characteristic chlorosis in leaves to a necrosis and large inhibition of growth. The severity of these abnormalities reflects the levels of yeast Pab1p expression in the transgenic plants. In contrast, no obvious differences could be seen in callus cultures between the transgene and vector control. Plants that display PAB-associated abnormalities were resistant to a range of plant pathogens, and had elevated levels of expression of a pathogenesis-related gene. These two properties – impairment of growth and induction of defense responses – indicate that the yeast PAB1 gene can act as a disease lesion mimic gene in plants.     

Inducible protein expression in <Emphasis Type="Italic">Drosophila</Emphasis> Schneider 2 cells using the <Emphasis Type="Italic">lac</Emphasis> operator–repressor system

Schneider line 2 cells, derived from Drosophila melanogaster, can be used as a highly versatile gene expression system. Two powerful promoters derived from the actin5C (Ac5) and metallothionein (Mtn) genes are available. The Mtn promoter can be used for the inducible expression of heterologous proteins unsuitable for constitutive expression. However, to circumvent using CuSO₄ or CdCl₂ as inducers of the Mtn promoter, we created a modified Ac5 promoter, Ac5LacO, in which two short lac operator sequences are embedded. Expression from the Ac5LacO promoter was regulated with co-expression of the lac repressor and IPTG. More than 25-fold induction of firefly luciferase expression was achieved in transient transfection experiments. Furthermore, we demonstrated that the lac operator–repressor regulatory system functioned in chromosomally integrated cell lines.     

Evidence that the geneuvrB is indispensable for a polymerase I deficient strain ofEscherichia coli K-12

Summary The gene coding for bacteriophage Lambda repressor (cI gene) has been fused to the lac operon of Escherichia coli. In some of the fusions Lambda repressor synthesis can be controlled by the lac operator and promoter. Upon induction of the lac operon the amount of Lambda repressor is increased by a factor of 7 over that found in a single lysogen. In combination with the polarity suppressor suA the induction factor rises to 20. Transducing phages of one fusion were constructed. After thermal induction of this phage the final level of Lambda repressor was enhanced by a factor of 150.Abbreviations xgal 5-bromo-4-chloro-3-indolyl--D-galactoside - IPTG isopropyl-thio--D-galactoside     

Regulated autocrine growth of CHO cells

The goal of this work was to engineer a CHO cell line capable ofautocrine growth in a fully defined protein-free medium. Thiswas accomplished by stable integration of the genes encodinginsulin-like growth factor I (IGF-I) and transferrin into thegenome of a CHO-K1 cell line. Thelac operator/repressorsystem was used to regulate the expression of the IGF-I gene with thelac operator sequence being placed upstream ofthe coding sequence for IGF-I. The expression of thelacrepressor protein was driven by a modified metallothioneinpromoter allowing repressor expression to be regulated by theculture medium. The cell line calledSuper CHO^r (r for regulated) was able to grow in protein-free medium in an autocrine fashion with a doubling time of 20–24 hr,either attached to microcarriers or as aggregate suspensioncultures. Upon addition of metal to the culture medium, therepressor protein was produced and bound to the operatorsequences shutting down the expression of IGF-I and arrestingthe growth of the cells. Expression of the human growth hormone(hGH) gene and production of hGH was induced by the presence ofmetal ions. It was possible to release the cells from growtharrest in the presence of metal by the addition of isopropyl-D-thiogalactopyranoside (IPTG), which prevented bindingof the repressor to its operator sequences. The ability to growCHO cells in fully defined protein-free medium and to be able toregulate their growth rate offers a number of advantages for theuse of these cells as hosts for the production of recombinantDNA derived proteins.     

Deletions fusing the i and lac regions of the chromosome in E. coli: isolation and mapping

Summary Three hundred and fifty deletions ending within the i gene (which codes for the lac repressor) at one terminus and within the z gene at the other terminus have been isolated. Twenty-nine of these have been mapped in detail and have endpoints which are distributed throughout the i gene. Two deletions which remove only the very end of the i gene still permit the synthesis of altered repressor molecules retaining some properties of the wildtype lac repressor. In strains carrying these deletions, the synthesis of lac permease is under the control of the i promoter. An additional 40 deletions have been found which apparently fuse the lac permease to an untranslated portion of the terminus of the i-message.     

Transgenic indica rice expressing Mirabilis jalapa antimicrobial protein (Mj-AMP2) shows enhanced resistance to the rice blast fungus Magnaporthe oryzae

Mj-AMP2, a knottin-type antimicrobial peptide, in vitro inhibits the growth of several plant pathogenic fungi including Magnaporthe oryzae. We demonstrate that transgenic rice (Oryza sativa L.) plants expressing the Mj-AMP2 gene show enhanced resistance to M. grisea, the causal agent of the rice blast disease. Mj-AMP2 was efficiently expressed and the level of Mj-AMP2 ranged from 0.32% to 0.38% of the total protein in the transgenic rice plants. In vitro inhibitory activity assays with the crude protein extract from transgenic rice indicated that the Mj-AMP2 protein produced was biologically active. Constitutive expression of Mj-AMP2 in transgenic rice reduces the growth of M. grisea by 63% with respect to untransformed control plant, and no effect on plant phenotype was observed. Transgene expression of Mj-AMP2 gene was not accompanied by an induction of pathogenesis-related (PR) gene expression indicating that the transgene product itself is directly active against the pathogen. The results presented in this study suggest that the Mj-AMP2 gene could be a useful candidate for protection of rice plants against the rice blast fungus M. grisea.     

Lac repressor binding to poly (d(A-T)). Conformational changes

The binding of $\text{lac}$ repressor to poly [d(A-T)] at low ionic strength has been investigated by circular dichroism, fluorescence and light scattering. Poly [d(A-T)] undergoes an important conformational change upon binding to $\text{lac}$ repressor. The maximum number of binding sites corresponds to about one tetrameric repressor per 11 base pairs of poly[d(A-T)]. The inducer isopropyl β-D-thiogalactoside (IPTG) does not affect the binding of $\text{lac}$ repressor to poly[d(A-T)]. It binds equally well to free and poly[d(A-T)] -bound repressor.     

Characterization of plant immunity-activating mechanism by a pyrazole derivative

ABSTRACT

A newly identified chemical, 4-{3-[(3,5-dichloro-2-hydroxybenzylidene)amino]propyl}-4,5-dihydro-1H-pyrazol-5-one (BAPP) was characterized as a plant immunity activator. BAPP enhanced disease resistance in rice against rice blast disease and expression of a defense-related gene without growth inhibition. Moreover, BAPP was able to enhance disease resistance in dicotyledonous tomato and Arabidopsis plants against bacterial pathogen without growth inhibition, suggesting that BAPP could be a candidate as an effective plant activator. Analysis using Arabidopsis sid2-1 and npr1-2 mutants suggested that BAPP induced systemic acquired resistance (SAR) by stimulating between salicylic acid biosynthesis and NPR1, the SA receptor protein, in the SAR signaling pathway.     

Identification of flowering genes in strawberry, a perennial SD plant

Background  

We are studying the regulation of flowering in perennial plants by using diploid wild strawberry (Fragaria vesca L.) as a model. Wild strawberry is a facultative short-day plant with an obligatory short-day requirement at temperatures above 15°C. At lower temperatures, however, flowering induction occurs irrespective of photoperiod. In addition to short-day genotypes, everbearing forms of wild strawberry are known. In 'Baron Solemacher' recessive alleles of an unknown repressor, SEASONAL FLOWERING LOCUS (SFL), are responsible for continuous flowering habit. Although flower induction has a central effect on the cropping potential, the molecular control of flowering in strawberries has not been studied and the genetic flowering pathways are still poorly understood. The comparison of everbearing and short-day genotypes of wild strawberry could facilitate our understanding of fundamental molecular mechanisms regulating perennial growth cycle in plants.     

Interaction of lac repressor with alternating poly d (A-T) and poly d (G-C). Circular dichroism studies

The binding of lac repressor to poly d(A-T) and poly d(G-C) has been studied using circular dichroism. The results indicate that the binding induces the same conformational change of both polynucleotides and perturbs the same number of nucleic acid bases (28 bases). It is shown that in 0.1 M phosphate buffer the CD measurement can be used to determine the binding constant of lac repressor to poly d(A-T). Competition experiments performed at various salt concentrations show that the stronger interaction of lac repressor for poly d(A-T) than for poly d(G-C) is based on difference in the dissociation rate of the complexes whereas the association rate for both polymers are similar.     

Tightly Regulated Expression Vectors for Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi, a bacterial species important in bioremediation and biopesticide degradation, are hindered by the lack of suitably regulated gene expression system. A tightly regulated gene-expression system was developed for O. anthropi using the lacI^q gene and a re-engineered coliphage T5 promoter containing completely symmetrical DNA segment that binds more efficiently to the lactose repressor. The β-galactosidase activity was increased 57-fold when the expression of the re-engineered T5 promoter was induced. The degree of induction was controllable by varying the concentration of inducer isopropyl-β-d-thiogalactopyranoside.     

COST1 balances plant growth and stress tolerance via attenuation of autophagy

ABSTRACT

In plants, macroautophagy/autophagy has been reported to function in various biotic and abiotic stress-response pathways, but few direct regulators linking stress and autophagy have yet been identified. Other than the conserved nutrient sensing kinase TOR (Target of Rapamycin), negative regulators that can directly modulate plant autophagy are unknown. We recently identified a mutant, termed cost1 (Constitutively Stressed 1), which has strong drought tolerance with constitutive induction of autophagy and broad expression of normally stress-responsive genes. The COST1 protein negatively regulates autophagy by direct interaction with the key autophagy adaptor ATG8E, thus directly linking autophagy and drought tolerance. Moreover, plant growth and development in a cost1 mutant is greatly retarded, suggesting that COST1 controls the tradeoff between growth and stress tolerance.