Expression of a bacterial gene in transgenic tobacco plants confers resistance to the herbicide 2,4-dichlorophenoxyacetic acid

Plants resistant to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) were produced through the genetic engineering of a novel detoxification pathway into the cells of a species normally sensitive to 2,4-D. We cloned the gene for 2,4-D monooxygenase, the first enzyme in the plasmid-encoded 2,4-D degradative pathway of the bacterium Alcaligenes eutrophus, into a cauliflower mosaic virus 35S promoter expression vector and introduced it into tobacco plants by Agrobacterium-mediated transformation. Transgenic tobacco plants expressing the highest levels of the monooxygenase enzyme exhibited increased tolerance to 2,4-D in leaf disc and seed germination assays, and young plants survived spraying with levels of herbicide up to eight times the usual field application rate. The introduction of the gene for 2,4-D monooxygenase into broad-leaved crop plants, such as cotton, should eventually allow 2,4-D to be used as an inexpensive post-emergence herbicide on economically important dicot crops.     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μ_max) of 0.158 h⁻¹. The half-maximum rate-associated substrate concentration (K_s) was 45 μM. At pH 8.5 μ_max was only 0.05 h⁻¹ and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μ_max with dichlorprop was around 0.2 h⁻¹ at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μ_max of 0.147 h⁻¹ and K_s of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h⁻¹ at pH 6.8 and up to D = 0.2 h⁻¹ at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.     

Effect of mouse calcineurin on induction and growth of rice callus transformed by the calcineurin gene

Calcineurin is a Ca²⁺/calmodulin dependent serine/threonine protein phosphatase, and has multiple functions in animal cells. In this work, mouse calcineurin was introduced into wild-type rice and the expression of calcineurin inhibited the induction and growth of rice calli. Inhibitor analysis showed that untransformed and CNAtr transgenic callus cultures had different sensitivity to cyclosporin A (CsA), a specific inhibitor of protein phosphatase calcineurin. When callus cultures were subject to 1 μM of CsA, the growth of calli induced from untransformed wild-type rice was inhibited. Interestingly, the growth inhibition of CNAtr transgenic calli was not detected in presence of 1 μM of CsA. Our findings showed that the heterologous calcineurin might be involved in the regulation of cell growth in plant cells.     

Starch fermentation by recombinant saccharomyces cerevisiae strains expressing the alpha-amylase and glucoamylase genes from lipomyces kononenkoae and saccharomycopsis fibuligera

Lipomyces kononenkoae and Saccharomycopsis fibuligera possess highly efficient alpha-amylase and/or glucoamylase activities that enable both of these yeasts to utilize raw starch as a carbon source. Eight constructs containing the L. kononenkoae alpha-amylase genes (LKA1 and LKA2), and the S. fibuligera alpha-amylase (SFA1) and glucoamylase (SFG1) genes were prepared. The first set of constructs comprised four single gene cassettes each containing one of the individual amylase coding sequences (LKA1, LKA2, SFA1 or SFG1) under the control of the phosphoglycerate kinase gene (PGK1) promoter and terminator, while the second set comprised two single cassettes containing SFA1 and SFG1 linked to their respective native promoters and terminators. The third set of constructs consisted of two double-gene cassettes, one containing LKA1 plus LKA2 under the control of the PGK1 promoter and terminator, and the other SFA1 plus SFG1 controlled by their respective native promoters and terminators. These constructs were transformed into a laboratory strain Saccharomyces cerevisiae (Sigma1278b). Southern-blot analysis confirmed the stable integration of the different gene constructs into the S. cerevisiae genome and plate assays revealed amylolytic activity. The strain expressing LKA1 and LKA2 resulted in the highest levels of alpha-amylase activity in liquid media. This strain was also the most efficient at starch utilization in batch fermentations, utilizing 80% of the available starch and producing 0.61g/100 mL of ethanol after 6 days of fermentation. The strain expressing SFG1 under the control of the PGK1 expression cassette gave the highest levels of glucoamylase activity. It was shown that the co-expression of these heterologous alpha-amylase and glucoamylase genes enhance starch degradation additively in S. cerevisiae. This study has resulted in progress towards laying the foundation for the possible development of efficient starch-degrading S. cerevisiae strains that could eventually be used in consolidated bioprocessing, and in the brewing, whisky, and biofuel industries.     

Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes

We have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick beta-actin mRNA and one oligonucleotide probe to chick alpha-cardiac actin mRNA. All the probes were 3' end-labeled with bio-11-dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin-alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of beta-actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of alpha-cardiac actin mRNA and the repression of beta-actin mRNA in differentiating myoblasts and in myotubes. With the alpha-cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When beta-actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of beta-actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. beta-Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both beta and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.     

Role of plasma membrane redox activities in elongation growth in plants

Comparing isolated plasma membrane vesicles and excised hypocotyl segments from etiolated seedlings of soybean [ Glycine max (L.) Merr. cv. Williams], certain antiproliferative agents that inhibited growth inhibited plasma membrane redox activities. Additionally, auxins that stimulated growth stimulated plasma membrane redox activities. Hormone stimulation was restricted to NADH oxidase (determined from disappearance of NADH) and was given both by isolated plasma membranes and by a soluhilizedenzyme preparation. Comparing IAA, the native auxin regulator, and 2,4-D, a synthetic regulator, stimulation was observed, hut the dose-response curves were different. Yet, the dose-response relationships of both stimulation of auxin growth and stimulation of NADH oxidase were parallel. Inhibition of auxin-induced growth by antiproliferative drugs was more complex. Some, like actinomycin D, preferentially inhibited NADH oxidase (EC 1.6.99.2) but inhibited NADH-ferricya-nide oxido-reductase (EC 1.6.99.3) as well. Others, like adriamycin, inhibited primarily the NADH-ferricyanide oxido-reductase. Therefore, growth control by auxin appeared to involve NADH oxidase as a rate-limiting terminal oxidase to link electron flow from NADH to oxygen. This observation may provide a fundamental difference from animal cells. With the latter, impermeant electron acceptors such as diferric transferrin or ferricyanide fulfill such a role. In plants, these impermeant electron acceptors were without effect on growth or were growth inhibitory.     

Improvement of cloned alpha-amylase gene expression in fed-batch culture of recombinant Saccharomyces cerevisiae by regulating both glucose and ethanol concentrations using a fuzzy controller

The effect of ethanol concentration on cloned gene expression in recombinant Saccharomyces cerevisiae strain 20B-12 containing one of two plasmids, pNA3 and pNA7, was investigated in batch cultures. Plasmids pNA3 and pNA7 contain the alpha-amylase gene under the control of the SUC2 or PGK promoter, respectively. When the ethanol concentration was controlled at 2 to 5 g/L, the gene expressions were two times higher than those at 20 g/L ethanol. The increase the gene expression by maintaining both the ethanol and glucose concentrations at low levels, a fuzzy ontroller was developed. The concentrations of glucose and ethanol were controlled simultaneously at 0.15 and 2 g/L, respectively, in the production phase using the fuzzy controller in fed-batch culture. The synthesis of alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase was induced by the low glucose concentration and maintained at a high level of activity by regulating the ethanol concentration at 2 g/L. The secretory alpha-amylase activities of cells harboring plasmids pNA3 and pNA7 in fed-batch culture were 175 and 395 U/mL, and their maximal specific activities 7.7 and 12.4 U/mg dry cells, respectively. These values are two to three times higher in activity and three to four times higher in specific activity than those obtained when glucose only was controlled. (c) 1994 John Wiley & Sons, Inc.     

Directed evolution of a bacterial alpha-amylase: toward enhanced pH-performance and higher specific activity

alpha-Amylases, in particular, microbial alpha-amylases, are widely used in industrial processes such as starch liquefaction and pulp processes, and more recently in detergency. Due to the need for alpha-amylases with high specific activity and activity at alkaline pH, which are critical parameters, for example, for the use in detergents, we have enhanced the alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild-type BAA and the mutants BAA S201N and BAA N297D were subjected to error-prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH range than the wild type, resulting in a 5-fold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4- and 1.5-fold, respectively. Another mutant (BAA 29) possesses a wild-type-like pH profile but possesses a 40-fold higher activity in periplasmic extracts and a 9-fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P, and A230V. In addition, three further mutations were found K406R, N414S, and E356D, the latter being present in other bacterial alpha-amylases.     

Phenylalanine ammonia-lyase: Regulation of its induction,and its role in plant development

The properties of phenylalanine ammonia-lyase from higher plants and its position in phenylpropanoid metabolism are briefly reviewed. Emphasis is then     

Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses

BACKGROUND: The Sleeping Beauty (SB) transposon system is a non-viral vector system that can integrate precise sequences into chromosomes. We evaluated the SB transposon system as a tool for gene therapy of mucopolysaccharidosis (MPS) types I and VII. METHODS: We constructed SB transposon plasmids for high-level expression of human beta-glucuronidase (hGUSB) or alpha-L-iduronidase (hIDUA). Plasmids were delivered with and without SB transposase to mouse liver by rapid, high-volume tail-vein injection. We studied the duration of expressed therapeutic enzyme activity, transgene presence by PCR, lysosomal pathology by toluidine blue staining and cell-mediated immune response histologically and by immunohistochemical staining. RESULTS: Transgene frequency, distribution of transgene and enzyme expression in liver and the level of transgenic enzyme required for amelioration of lysosomal pathology were estimated in MPS I and VII mice. Without immunomodulation, initial GUSB and IDUA activities in plasma reached > 100-fold of wild-type (WT) levels but fell to background within 4 weeks post-injection. In immunomodulated transposon-treated MPS I mice plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of MPS I mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, some of which were CD4- or CD8-positive. CONCLUSIONS: Our results demonstrate the feasibility of achieving prolonged expression of lysosomal enzymes in the liver and reversing MPS disease in adult mice with a single dose of therapeutic SB transposons.     

Selection for increased vernolate tolerance in tobacco (Nicotiana tabacum L.) cell cultures

Vernolate (S-propyl N, N-dipropylthiocarbamate) has been used to select thiocarbamate-tolerant tobacco cell lines. Cross-tolerance to other thiocarbamate herbicides was observed. Tolerance was initially unstable in the absence of continued selection, but shoot regeneration produced plants in which some tolerance was stably expressed. Morphological abnormalities in the regenerated plants are described.     

Synthesis and secondary structure of oligo(alpha-isobutyl beta,L-aspartate)s

The oligo(beta-peptide)s, hexa(alpha-isobutyl beta,L-aspartate) (Hex-AIBLA) and octa(alpha-isobutyl beta,L-aspartate) (Oct-AIBLA), were synthesized in solution by using standard coupling methods. Powder x-ray diffraction showed that the octamer crystallized in the two helical crystal forms known to exist in the homologous poly(beta-peptide), whereas the hexamer seemed to adopt an extended conformation. Both CD and 1H-NMR spectra of Oct-AIBLA in MeOH revealed the presence of a regularly folded conformation in this solvent, presumably the 3(14) helix. The helix-to-coil transition of Oct-AIBLA was observed to take place upon heating in both MeOH and CHCl3, in the second case associated with a not-well-defined aggregation-disaggregation process. The spectroscopic evidence obtained on the presence of folded structures in Hex-AIBLA were much weaker than for the octamer.     

Effects of Ginkgo biloba extract on the proliferation of vascular smooth muscle cells in vitro and on intimal thickening and interleukin-1beta expression after balloon injury in cholesterol-fed rabbits in vivo

Restenosis may develop in response to cytokine activation and smooth muscle cell proliferation. Ginkgo biloba extract (EGb) has been used to treat cardiovascular and cerebrovascular diseases. In the present study, the effects of EGb on the growth of cultured vascular smooth muscle cells (VSMC), as well as on the expression of interleukin-1beta (IL-1beta) and the intimal response in balloon-injured arteries of cholesterol-fed rabbits, were investigated. Using bromodeoxyuridine incorporation as an index of cell proliferation, EGb was found to inhibit serum-induced mitogenesis of cultured rat aorta VSMC in a dose-dependent manner. In vivo, EGb and probucol ( positive control) reduced the atheroma area in thoracic aortas of male New Zealand white rabbits fed a 2% cholesterol diet for 6 weeks with balloon denudation of the abdominal aorta being performed at the end of the third week. Intimal hyperplasia, expressed as the intimal/medial area ratio, in the abdominal aortas was significantly inhibited in the both the EGb group (0.61 +/- 0.06) and the probucol group (0.55 +/- 0.03) compared to the C group (0.87 +/- 0.02). In the balloon-injured abdominal aorta, both EGb and probucol significantly reduced IL-1beta mRNA and protein expression and the percentage of proliferating cells. The inhibitory effects of EGb on the intimal response might be attributed to its antioxidant capacity. EGb may have therapeutic potential for the prevention of restenosis after angioplasty.     

Comparison of three different promoter systems for secretory alpha-amylase production in fed-batch cultures of recombinant Saccharomyces cerevisiae

Cloned gene expression in recombinant Saccharomyces cerevisiae 20B-12 containing three different plasmids was compared in batch and fed-batch cultures. The plasmids pNA3, pNA7, and pNA9 contain the alpha-amylase gene under the control of SUC2, PGK, and GAL7 Promoters, respectively. The synthesis of alpha-amylase was therefore induced by low glucose concentration for the SUC2 and PGK promoters, and by galactose for GAL7 promoter. The specific cell growth rates were similar among cells harboring the three different plasmids; they decreased from 0.35 to 0.38 h(-1) during the cell growth phase to 0.03 to 0.06h(-1) during the production phase. The secretory alpha-amylase activity of cells harboring plasmid pNA7 was 129 U/mL in fed-batch culture, which was 1.4 and 2 times as high as the activities of cells harboring plasmids pNA3 and pNA9, respectively. The secretion ratios (amount of extracellular alpha-amylase activity/amounts of total alpha-amylase activity) of cells harboring plasmids pNA3, pNA7, and pNA9 were 91.4%, 94.5%, and 95.3%, respectively. (c) 1993 Wiley & Sons, Inc.