Nuclear male sterility induced by pollen-specific expression of a ribonuclease

The promoter of a rice pollen-specific gene, PS1, has been fused to the Bacillus amyloliquefaciens Barnase gene which encodes a secreted ribonuclease. The PS1-Barnase chimeric gene has been introduced into tobacco. These transgenic tobacco plants show normal vegetative and floral development, but they display a range of reproductive properties from slightly reduced in fertility to completely sterile. Barnase mRNAs are detectable in the pollen from transgenic plants which do not show an obvious fertility-related phenotype, and in a few plants which have a mildly reduced-fertile phenotype. However, transgenic plants with a severely reduced-fertile or sterile phenotype do not accumulate detectable amounts of Barnase mRNA in their pollen, and the quality of their RNA is poor, presumably because of extensive RNA degradation. Reciprocal crosses between these transgenic plants and wild-type controls showed that the reduced-fertile phenotype is associated only with the transgenic pollen. When used as the female parent, these PS1-Barnase transgenic plants are fully fertile. Anthers in the severely sterile transgenic plants develop normally, but the majority of their pollen grains have abnormal morphology and they fail to germinate. These results indicate that expression of a pollen-specific cytotoxic gene induces lethality in pollen and may lead to severely reduced male fertility.     

Ordered differential display: a simple method for systematic comparison of gene expression profiles.

A method for display of 3'-end restriction fragments of cDNAs is proposed, extending the idea reported recently. First, representative pools of such fragments are selectively amplified using PCR suppression effect. Then, simplified subsets of these fragments suitable for comparison by PAGE are amplified by adapter-specific primers extended by two randomly picked bases at their 3'ends. By testing all possible combinations of extended primers the whole mRNA pool may be systematically investigated. The method was applied to search for molecular regional markers of freshwater planarian Dugesia tigrina .     

FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing

Massively parallel sequencing of cell-free, maternal plasma DNA was recently demonstrated to be a safe and effective screening method for fetal chromosomal aneuploidies. Here, we report an improved sequencing method achieving significantly increased throughput and decreased cost by replacing laborious sequencing library preparation steps with PCR employing a single primer pair designed to amplify a discrete subset of repeated regions. Using this approach, samples containing as little as 4% trisomy 21 DNA could be readily distinguished from euploid samples.     

Enhanced quantitative resistance to Leptosphaeria maculans conferred by expression of a novel antimicrobial peptide in canola (Brassica napus L.)

The novel antimicrobial peptide MiAMP1, originally isolated from the seeds of Macadamia integrifolia, was constitutively expressed in transgenic tobacco and canola plants to test its effect on disease resistance. Analysis of plants transformed with 35S-MiAMP1 construct by northern and western blot analyses demonstrated the presence of MiAMP1 mRNA and the mature peptide in the transgenic plants. The MiAMP1 purified from the leaves of transgenic plants was biologically active with the same in vitro antifungal activity as native MiAMP1 purified from the seeds of macadamia. The effect of MiAMP1 expression on the economically important canola pathogen Leptosphaeria maculans (causal agent of blackleg disease) was evaluated in comparison with an untransformed control line and an azygous segregant derived from one of the transgenic lines. Lesion development on the cotyledons of the inoculated canola seedlings was significantly reduced in the T₂ progeny of seven independently transformed transgenic lines. These results suggested that, transgenic canola expressing MiAMP1 may be useful for the management of blackleg disease.     

Twin PCRs: a simple and efficient method for directional cloning of PCR products

A simple and efficient method was developed for directional cloning of PCR products without any restriction enzyme digestion of the amplified sequence. Two pairs of primers were designed in which parts of two restriction enzyme recognition sequences were integrated, and the primers were used for two parallel PCRs. The PCR products were mixed, heat denatured and re-annealed to generate hybridized DNA fragments bearing sticky ends compatible with restriction enzymes. This method is particularly useful when it is necessary to use a restriction enzyme but there is an additional internal restriction site within the amplified sequence, or when there are problems caused by end sensitivity of restriction enzymes.     

Construction of an efficient expression system for Aspergillus isopullulanase in Pichia pastoris,and a simple purification method

Aspergillus niger ATCC 9642 isopullulanase (IPU) was heterologously expressed by Pichia pastoris GS115 under three different signal sequences of Saccharomyces cerevisiae acid phosphatase, S. cerevisiae alpha-factor prepro peptide, and A. niger isopullulanase. One-step purification using lectin Con A affinity chromatography yielded recombinant IPU (IPU-PP) with high purity. IPU-PP had a higher carbohydrate content than native IPU and IPU-AO expressed in A. oryzae M-2-3. IPU-PP hydrolyzed various substrates containing the structure of panose, which indicated a strict subsite recognition of the panose motif.     

The investigation of the combined effects of gamma irradiation and environmental manipulation on mortality and sterility of Rhyzopertha dominica (Fabricius) (Coleoptera: Bostrichidae)

With respect to the limitations use of methyl bromide and phosphine, employing ionizing radiation to control stored product pests has attracted great attention. The aim of this study is the investigation of the combined effects of gamma irradiation as viable alternatives to synthetic insecticides and environmental management on mortality and sterility of Rhyzopertha dominica (Fabricius) in a wheat cultivar (Gascogen). The effect of doses range from 30 to 2000?Gy gamma irradiation in combination with manipulation of temperature (15, 21, 27 and 32?°C) and relative humidity (20%, 50%, 65% and 85%) on 5–10?days old adults of R. dominica in Gascogen cultivar of wheat were explored. The experiments were repeated three times and conducted in The Nuclear Agriculture Research School in Karaj and laboratory of shahid steki silo in shahre-kord. Probit analysis revealed that both temperature and relative humidity had combined effects when used with gamma irradiation. The lowest doses of gamma ray required to kill 25% (14.2?Gy) and 50% (610.8?Gy) of the population (LD₂₅ and LD₅₀) were recorded at 21?°C and 85% relative humidity respectively. The low dose for 99% mortality (LD₉₉; 2386.7?Gy) was recorded for beetles maintained at 21?°C and 50% relative humidity. The effect of temperature (15, 21, 27 and 32?°C) on sterility caused by gamma irradiation was also investigated. The results showed that the F₁ generation emerged only when the beetles were treated with doses of 0–100?Gy at 32?°C and 0–70?Gy at 27?°C. These results indicate that temperature and relative humidity play an important role in the susceptibility of the lesser grain beetle to gamma irradiation. The results suggest that controlling the efficiency of gamma radiation through environmental control allows the use of low doses of gamma radiation that have a less harmful effect on human health, non-target organisms and seed agronomic features.     

On fluctuation analysis: a new,simple and efficient method for computing the expected number of mutants

Fluctuation analysis, which is often used to demonstrate random mutagenesis in cell lines (and to estimate mutation rates), is based on the properties of a probability distribution known as the Luria-Delbrück distribution (and its generalizations). The two main new results reported in this paper are (i) a simple, completely general, and computationally efficient procedure for calculating probability distributions arising from fluctuation analysis and (ii) the formula for this procedure when cells in a colony have only grown for a finite number of generations after initial seeding. It is also shown that the procedure reduces to one that was developed earlier when an infinite number of generations is assumed. The derivation of the generating function of the distribution is also clarified. The results obtained should also be useful to experimentalists when only a relatively short time elapses between seeding and harvestint cultures for fluctuation analysis.     

Preparation of 18F-human serum albumin: a simple and efficient protein labeling method with 18F using a hydrazone-formation method

18F-labeling of proteins and peptides is important for positron emission tomography (PET). Although there are many methods for the labeling of proteins with (18)F, most of these are characterized by complicated procedures or low yields. Here, we report a novel and simple method which includes the preparation of [18F]fluorobenzaldehyde ([18F]FBA) and successive conjugation with hydrazinonicotinic acid-human serum albumin conjugate (HYNIC-HSA) via hydrazone formation. HYNIC-HSA, which can also be used for labeling with (99m)Tc, was prepared via reaction with N-hydroxysuccinimide (NHS) or tetrafluorophenyl (TFP) esters of HYNIC with HSA. No-carrier-added [18F]FBA was prepared by the nucleophilic substitution of [18F]fluoride to 4-trimethylammonium benzaldehyde triflate in the presence of tetrabutylammonium bicarbonate. [18F]FBA was purified by passing ion exchange cartridges (IC-H and QMA) and was adsorbed to a C18 Sep-Pak cartridge. The adsorbed [18F]FBA was then eluted with 50% ethanol. HYNIC-HSA was added to the solution and conjugated with [18F]FBA via hydrazone formation. 18F-HSA was purified with a PD10 column. Biodistribution of 18F-HSA, (99m)Tc-HSA, and [18F]FBA in mice were investigated at 10, 20, and 60 min after intravenous injection. The number of conjugated HYNIC molecules per HSA ranged from 5.2 to 23.2 depending on the reaction conditions. The labeling efficiency of 18F-FBA was 67 +/- 15.7%. The radiochemical purity after purification was over 99%. The conjugation efficiency of HYNIC-HSA with [18F]FBA was between 25% and 90%. The conjugation efficiency was observed to increase with increases in the number of conjugated HYNIC, the HYNIC-HSA concentration, or temperature. 18F-HSA exhibited a biodistribution pattern similar to that of (99m)Tc-HSA while [18F]FBA showed much lower blood activity than that of 18F-HSA and (99m)Tc-HSA. We concluded that 18F-HSA was successfully labeled using a novel method which involves hydrazone formation between [18F]FBA and HYNIC-HSA. This method can be applied to the 18F-labeling of other proteins or peptides.     

Field trial detects incomplete barstar attenuation of vegetative cytotoxicity in Populus trees containing a poplar LEAFY promoter::barnase sterility transgene

We tested the efficacy of an attenuation system developed to preclude the deleterious effects of floral promoter::cytotoxin genes on vegetative growth of transgenic sterile plants. We tested the promoter (2.6 kb 5′ region) of the poplar LEAFY gene PTLF driving barstar, combined on the same T-DNA with barstar driven by either the CaMV 35S basal promoter +5 to −72 fragment (35SBP), 35SBP fused to the TMV omega element (35SBP omega), or the NOS promoter. The unattenuated pPTLF::barnase construct failed to give rise to any transgenic events, suggesting substantial non-reproductive expression from this promoter. The barstar-attenuated constructs enabled transformation, but the rate was reduced by nearly one-third. Four events (7% of attenuated events) had highly abnormal morphology, and were identified during the early phases of propagation; these events had significantly higher barnase:barstar expression ratios based on quantitative RT-PCR. A greenhouse study showed that phenotypically normal attenuated plants grew at the same rate as wild-type and barnase-lacking transgenic plants. A statistically significant positive linear association was found between relative growth rate (RGR) and barstar:barnase ratio in the attenuated events, and graphical analysis suggested a threshold for barstar attenuation of barnase, above which additional levels of barstar did not provide further attenuation. Surprisingly, the appearance and growth rate of the nearly all of the attenuated events were substantially reduced after one or two growing seasons in the field, and the extent of growth reduction was associated with barstar:barnase expression ratio. These results demonstrate the importance of field testing during early phases of research to identify pleiotropic effects of transgenic sterility genes in trees.     

Biosynthesis of zearalenone: a simple and efficient method to incorporate [13C]acetate label by using solid cultures

A suitable method was developed to efficiently incorporate 13C-labeled acetate into zearalenone by using solid cultures. Periodic feeding of the label during the zearalenone production phase significantly increased the label incorporation for the singly labeled acetate.     

Biosynthesis of zearalenone: a simple and efficient method to incorporate [13C]acetate label by using solid cultures.

A suitable method was developed to efficiently incorporate 13C-labeled acetate into zearalenone by using solid cultures. Periodic feeding of the label during the zearalenone production phase significantly increased the label incorporation for the singly labeled acetate.     

A novel and simple method of screening compounds for interaction with DNA: A validation study

We report the development of a simple, cost-effective assay for detecting compounds that have the ability to interact with and modify DNA. Potential uses for the assay lie in the areas of early genotoxicity testing of drug candidates, anticancer and antibiotic drug discovery, environmental monitoring and testing in the food, beverage and cosmetics industries. At present the assay has been used to assess direct-acting compounds only and it is yet to be established whether the assay is compatible with bio-activation. The methodology is based on the oxidative reaction of potassium permanganate with pyrimidine bases, which have become perturbed and more reactive by the agent under test. Results are recorded by use of UV/vis spectroscopy. The adaptation to a multi-well plate format provides the capacity for high throughput utilizing small amounts of compounds. Over 100 compounds, comprising different classes of DNA-binding chemicals as well as non-binding controls, have been put through the assay and the results compared with existing genotoxicity testing data from other methods. The assay has shown to be predictive of the results of other genotoxicity testing methods. We have found that the method is overall predictive of 71% of Ames bacterial reverse-mutation test results (where data are given) encompassing both negative and positive results.     

Exposure reconstruction for reducing uncertainty in risk assessment: example using MTBE biomarkers and a simple pharmacokinetic model

Adverse health risks from environmental agents are generally related to average (long-term) exposures. Because a given individual's contact with a pollutant is highly variable and dependent on activity patterns, local sources and exposure pathways, simple 'snapshot' measurements of surrounding environmental media may not accurately assign the exposure level. Furthermore, susceptibility to adverse effects from contaminants is considered highly variable in the population so that even similar environmental exposure levels may result in differential health outcomes in different individuals. The use of biomarker measurements coupled to knowledge of rates of uptake, metabolism and elimination has been suggested as a remedy for reducing this type of uncertainty. To demonstrate the utility of such an approach, we invoke results from a series of controlled human exposure tests and classical first-order rate kinetic calculations to estimate how well spot measurements of methyl tertiary butyl ether and the primary metabolite, tertiary butyl alcohol, can be expected to predict different hypothetical scenarios of previous exposures. We found that blood and breath biomarker measurements give similar results and that the biological damping effect of the metabolite production gives more stable estimates of previous exposure. We also explore the value of a potential urinary biomarker, 2-hydroxyisobutyrate suggested in the literature. We find that individual biomarker measurements are a valuable tool in reconstruction of previous exposures and that a simple pharmacokinetic model can identify the time frames over which an exogenous chemical and the related chemical biomarker are useful. These techniques could be applied to broader ranges of environmental contaminants to assess cumulative exposure risks if ADME (Absorption, Distribution, Metabolization and Excretion) is understood and systemic biomarkers can be measured.     

Exposure reconstruction for reducing uncertainty in risk assessment: example using MTBE biomarkers and a simple pharmacokinetic model

Adverse health risks from environmental agents are generally related to average (long-term) exposures. Because a given individual's contact with a pollutant is highly variable and dependent on activity patterns, local sources and exposure pathways, simple ‘snapshot’ measurements of surrounding environmental media may not accurately assign the exposure level. Furthermore, susceptibility to adverse effects from contaminants is considered highly variable in the population so that even similar environmental exposure levels may result in differential health outcomes in different individuals. The use of biomarker measurements coupled to knowledge of rates of uptake, metabolism and elimination has been suggested as a remedy for reducing this type of uncertainty. To demonstrate the utility of such an approach, we invoke results from a series of controlled human exposure tests and classical first-order rate kinetic calculations to estimate how well spot measurements of methyl tertiary butyl ether and the primary metabolite, tertiary butyl alcohol, can be expected to predict different hypothetical scenarios of previous exposures. We found that blood and breath biomarker measurements give similar results and that the biological damping effect of the metabolite production gives more stable estimates of previous exposure. We also explore the value of a potential urinary biomarker, 2-hydroxyisobutyrate suggested in the literature. We find that individual biomarker measurements are a valuable tool in reconstruction of previous exposures and that a simple pharmacokinetic model can identify the time frames over which an exogenous chemical and the related chemical biomarker are useful. These techniques could be applied to broader ranges of environmental contaminants to assess cumulative exposure risks if ADME (Absorption, Distribution, Metabolization and Excretion) is understood and systemic biomarkers can be measured.     

Transfection of Schistosoma mansoni by electroporation and the description of a new promoter sequence for transgene expression

We sought to investigate the efficacy of electroporation for the introduction of plasmid-based DNA constructs into Schistosoma mansoni, and expanded our study to examine parameters governing transgene expression, including requirements of a 5′ and 3′ flanking sequence, as well as parasite developmental effects on transgene expression. We used luciferase as a reporter gene for this application. Our data show that electroporation allows the transfection of immature schistosomes, and defines 5′ promoter sequence from the schistosome actin gene (SmAct1.1), coupled promiscuously with various 3′ terminator sequences, as a powerful promoter of transgene expression in growing, but not early non-growing, schistosomula. The methodology described herein will facilitate ectopic expression of genes of interest in schistosomes.     

Proteomics reveals differential expression of proteins related to a variety of metabolic pathways by genistein-induced Bradyrhizobium japonicum strains

The rhizobia-legume symbiosis requires a coordinated molecular interaction between the symbionts, initiated by seed and root exudation of several compounds, mainly flavonoids, that trigger the expression of nodulation genes in the bacteria. Since the role of flavonoids seems to be broader than the induction of nodulation genes, we aimed at characterizing genistein-induced proteins of Bradyrhizobium japonicum CPAC 15 (= SEMIA 5079), used in commercial soybean inoculants in Brazil, and of two genetically related strains grown in vitro. Whole-cell proteins were extracted both from induced (1 μM genistein) and from non-induced cultures of the three strains, and separated by two-dimensional electrophoresis. Spot profiles were compared between the two conditions and selected spots were excised and identified by mass spectrometry. Forty-seven proteins were significantly induced by genistein, including several hypothetical proteins, the cytoplasmic flagellar component FliG, periplasmic ABC transporters, a protein related to biosynthesis of exopolysaccharides (ExoN), and proteins involved in redox-state maintenance. Noteworthy was the induction of the PhyR-σ^EcfG regulon, recently demonstrated to be involved in the symbiotic efficiency of, and general stress response in B. japonicum. Our results confirm that the role of flavonoids, such as genistein, can go far beyond the expression of nodulation-related proteins in B. japonicum.