Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro

Transcriptional gene silencing (TCS) is often associated with an increased level of cytosine methylation in the affected promoters. The effect of methylation of the cauliflower mosaic virus (CaMV) 35S promoter sequence on its binding to factors present in the nuclei was analyzed by electrophoretic mobility shift assays using extracts of petunia flowers. Specific DNA-protein interactions were detected in the region of the CaMV 35S promoter that contains the as-1 element and the region between -345 and -208. The binding of protein factor(s) to the as-1 element was influenced by cytosine methylation, whereas the binding to the region between -345 and -208 was unaffected. The results suggest that cytosine methylation of the as-1 element potentially affects the activity of the CaMV 35S promoter. © Georg Thieme Verlag KG Stuttgart.

Ecological risk analysis of transgenic plants

Public concern about the safety of many forms of industrial technology are known to be linked to a range of factors including a perceived lack of confidence in regulatory decision making.1 The use of transgenic plants in agriculture may be seen as an issue that could generate similar concern. Criticism has been made about the completeness of knowledge on the potential for aberrant behaviour of genetically manipulated organisms (GMO's) in release environments, and the adequacy of existing pre­‐release screening and assessment methodologies (Goldberg & Tjaden, 1990). Such comments are important because any perceived shortcomings in the pre-release assessment of GMO safety may lead to decreased public support of the technology -­‐and the industry itself...

Bimolecular interaction of Insulin-Like Growth Factor (IGF) binding protein-2 with αvβ3 negatively modulates IGF-I-Mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the αvβ3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and αvβ3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7β3, which overexpressed the αvβ3 integrin. Collectively, these results indicate that αvβ3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.

Binding and degradation of hyaluronan by human breast cancer cell lines expressing different forms of CD44 : correlation with invasive potential

In the present study, we examined a panel of human breast cancer cell lines with regard to their expression of CD44 and ability to bind and degrade hyaluronan. The cell lines expressed varying amounts of different molecular weight forms of CD44 (85-200 kDa) and, in general, those that expressed the greatest amounts of CD44 were the most invasive as judged by in vitro assays. In addition, the ability to bind and degrade hyaluronan was restricted to the cell lines expressing high levels of CD44, and both these functions were blocked by an antibody to CD44 (Hermes-1). Moreover, the rate of [3H]hyaluronan degradation was highly correlated with the amount of CD44 (r = 0.951, P < 0.0001), as well as with the invasive potential of the cells. Scatchard analysis of the [3H]hyaluronan binding of these cells revealed the existence of significant differences in both their binding capacity and their dissociation constant. To determine the source of this deviation, the different molecular weight forms of CD44 were partially separated by gel filtration chromatography. In all cell lines, the 85 kDa form was able to bind hyaluronan, although with different affinities. In contrast, not all of the high molecular weight forms of CD44 had this ability. These results illustrate the diversity of CD44 molecules in invasive tumor cells, and suggest that one of their major functions is to degrade hyaluronan.

Secreted products and extracellular matrix from testicular peritubular myoid cells influence androgen-binding protein secretion by Sertoli cells in culture

Metabolic cooperation mediated by secreted factors between Sertoli cells and peritubular myoid cells has been well documented. We have confirmed that factors secreted by peritubular myoid cells modulate androgen-binding protein (ABP) secretion by Sertoli cells and shown further that this can also be achieved with peritubular myoid cell extracellular matrix (ECM). While peritubular myoid cell ECM potentiated the stimulatory effect of dibutyryl cyclic AMP on Sertoli cell ABP secretion, secreted factors did not, suggesting that the two components influence Sertoli cells through distinct mechanisms. We also tested other factors and other cell lines for effects on ABP production by Sertoli cells. The addition of human plasma fibronectin or conditioned medium from the basement membrane-producing Englebreth-Holm- Swarm sarcoma also stimulated ABP secretion by Sertoli cells. Cocultures of epithelial Sertoli cells with the cells of mesenchymal origin, such as testicular peritubular myoid cells, embryonic skin fibroblasts, and bladder smooth muscle cells, significantly stimulated ABP secretion by Sertoli cells, but co-culture with the epithelial-derived Martin-Darby canine kidney cell line had no effect on Sertoli cell-secreted ABP levels. Our data further define the epithelial-mesenchymal cell interaction that exists between Sertoli cells and peritubular myoid cells in the mammalian testis.

Hydrolysis of triasulfuron, metsulfuron-methyl and chlorsulfuron in alkaline soil and aqueous solutions

The hydrolysis of triasulfuron, metsulfuron-methyl and chlorsulfuron in aqueous buffer solutions and in soil suspensions at pH values ranging from 5.2 to 11.2 was investigated. Hydrolysis of all three compounds in both aqueous buffer and soil suspensions was highly pH-sensitive. The rate of hydrolysis was much faster in the acidic pH range (5.2-6.2) than under neutral and moderately alkaline conditions (8.2-9.4), but it increased rapidly as the pH exceeded 10.2. All three compounds degraded faster at pH 5.2 than at pH 11.2. Hydrolysis rates of all three compounds could be described well with pseudo-first-order kinetics. There were no significant differences (P =0.05) in the rate constants (k, day-1) of the three compounds in soil suspensions from those in buffer solutions within the pH ranges studied. A functional relationship based on the propensity of nonionic and anionic species of the herbicides to hydrolyse was used to describe the dependence of the 'rate constant' on pH. The hydrolysis involving attack by neutral water was at least 100-fold faster when the sulfonylurea herbicides were undissociated (acidic conditions) than when they were present as the anion at near neutral pH. In aqueous buffer solution at pH > 11, a prominent degradation pathway involved O-demethylation of metsulfuron-methyl to yield a highly polar degradate, and hydrolytic opening of the triazine ring. It is concluded that these herbicides are not likely to degrade substantially through hydrolysis in most agricultural (C) 2000 Society of Chemical Industry.

Impact of a change in tillage and crop residue management practice on soil chemical and microbiological properties in a cereal-producing red duplex soil in NSW, Australia

The effect of a change of tillage and crop residue management practice on the chemical and micro-biological properties of a cereal-producing red duplex soil was investigated by superimposing each of three management practices (CC: conventional cultivation, stubble burnt, crop conventionally sown; DD: direct-drilling, stubble retained, no cultivation, crop direct-drilled; SI: stubble incorporated with a single cultivation, crop conventionally sown), for a 3-year period on plots previously managed with each of the same three practices for 14 years. A change from DD to CC or SI practice resulted in a significant decline, in the top 0-5 cm of soil, in organic C, total N, electrical conductivity, NH4-N, NO3-N, soil moisture holding capacity, microbial biomass and CO2 respiration as well as a decline in the microbial quotient (the ratio of microbial biomass C to organic C; P <0.05). In contrast, a change from SI to DD or CC practice or a change from CC to DD or SI practice had only negligible impact on soil chemical properties (P >0.05). However, there was a significant increase in microbial biomass and the microbial quotient in the top 0-5 cm of soil following the change from CC to DD or SI practice and with the change from SI to DD practice (P <0.05). Analysis of ester-linked fatty acid methyl esters (EL-FAMEs) extracted from the 0- to 5-cm and 5- to 10-cm layers of the soils of the various treatments detected changes in the FAME profiles following a change in tillage practice. A change from DD practice to SI or CC practice was associated with a significant decline in the ratio of fungal to bacterial fatty acids in the 0- to 5-cm soil (P <0.05). The results show that a change in tillage practice, particularly the cultivation of a previously minimum-tilled (direct-drilled) soil, will result in significant changes in soil chemical and microbiological properties within a 3-year period. They also show that soil microbiological properties are sensitive indicators of a change in tillage practice.

Statistical analysis of reduction in tensile strength of cotton strips as a measure of soil microbial activity

The cotton strip assay (CSA) is an established technique for measuring soil microbial activity. The technique involves burying cotton strips and measuring their tensile strength after a certain time. This gives a measure of the rotting rate, R, of the cotton strips. R is then a measure of soil microbial activity. This paper examines properties of the technique and indicates how the assay can be optimised. Humidity conditioning of the cotton strips before measuring their tensile strength reduced the within and between day variance and enabled the distribution of the tensile strength measurements to approximate normality. The test data came from a three-way factorial experiment (two soils, two temperatures, three moisture levels). The cotton strips were buried in the soil for intervals of time ranging up to 6 weeks. This enabled the rate of loss of cotton tensile strength with time to be studied under a range of conditions. An inverse cubic model accounted for greater than 90% of the total variation within each treatment combination. This offers support for summarising the decomposition process by a single parameter R. The approximate variance of the decomposition rate was estimated from a function incorporating the variance of tensile strength and the differential of the function for the rate of decomposition, R, with respect to tensile strength. This variance function has a minimum when the measured strength is approximately 2/3 that of the original strength. The estimates of R are almost unbiased and relatively robust against the cotton strips being left in the soil for more or less than the optimal time. We conclude that the rotting rate X should be measured using the inverse cubic equation, and that the cotton strips should be left in the soil until their strength has been reduced to about 2/3.

Using the Gini coefficient with BIOLOG substrate utilisation data to provide an alternative quantitative measure for comparing bacterial soil communities

A measure quantifying unequal use of carbon sources, the Gini coefficient (G), has been developed to allow comparisons of the observed functional diversity of bacterial soil communities. This approach was applied to the analysis of substrate utilisation data obtained from using BIOLOG microtiter plates in a study which compared decomposition processes in two contrasting plant substrates in two different soils. The relevance of applying the Gini coefficient as a measure of observed functional diversity, for soil bacterial communities is evaluated against the Shannon index (H) and average well colour development (AWCD), a measure of the total microbial activity. Correlation analysis and analysis of variance of the experimental data show that the Gini coefficient, the Shannon index and AWCD provided similar information when used in isolation. However, analyses based on the Gini coefficient and the Shannon index, when total activity on the microtiter plates was maintained constant (i.e. AWCD as a covariate), indicate that additional information about the distribution of carbon sources being utilised can be obtained. We demonstrate that the Lorenz curve and its measure of inequality, the Gini coefficient, provides not only comparable information to AWCD and the Shannon index but when used together with AWCD encompasses measures of total microbial activity and absorbance inequality across all the carbon sources. This information is especially relevant for comparing the observed functional diversity of soil microbial communities.

Capacity of fatty acid profiles and substrate utilization patterns to describe differences in soil microbial communities associated with increased salinity or alkalinity at three locations in South Australia

Fatty acid methyl ester (FAME) profiles, together with Biolog substrate utilization patterns, were used in conjunction with measurements of other soil chemical and microbiological properties to describe differences in soil microbial communities induced by increased salinity and alkalinity in grass/legume pastures at three sites in SE South Australia. Total ester-linked FAMEs (EL-FAMEs) and phospholipid-linked FAMEs (PL-FAMEs), were also compared for their ability to detect differences between the soil microbial communities. The level of salinity and alkalinity in affected areas of the pastures showed seasonal variation, being greater in summer than in winter. At the time of sampling for the chemical and microbiological measurements (winter) only the affected soil at site 1 was significantly saline. The affected soils at all three sites had lower organic C and total N concentrations than the corresponding non-affected soils. At site 1 microbial biomass, CO 2-C respiration and the rate of cellulose decomposition was also lower in the affected soil compared to the non-affected soil. Biomarker fatty acids present in both the EL- and PL-FAME profiles indicated a lower ratio of fungal to bacterial fatty acids in the saline affected soil at site 1. Analysis of Biolog substrate utilization patterns indicated that the bacterial community in the affected soil at site 1 utilized fewer carbon substrates and had lower functional diversity than the corresponding community in the non-affected soil. In contrast, increased alkalinity, of major importance at sites 2 and 3, had no effect on microbial biomass, the rate of cellulose decomposition or functional diversity but was associated with significant differences in the relative amounts of several fatty acids in the PL-FAME profiles indicative of a shift towards a bacterial dominated community. Despite differences in the number and relative amounts of fatty acids detected, principal component analysis of the EL- and PL-FAME profiles were equally capable of separating the affected and non-affected soils at all three sites. Redundancy analysis of the FAME data showed that organic C, microbial biomass, electrical conductivity and bicarbonate-extractable P were significantly correlated with variation in the EL-FAME profiles, whereas pH, electrical conductivity, NH 4-N, CO 2-C respiration and the microbial quotient were significantly correlated with variation in the PL-FAME profiles. Redundancy analysis of the Biolog data indicated that cation exchange capacity and bicarbonate-extractable K were significantly correlated with the variation in Biolog substrate utilization patterns.

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.

Human single-stranded DNA binding protein 1 (hSSB1/NABP2) is required for the stability and repair of stalled replication forks

Aberrant DNA replication is a primary cause of mutations that are associated with pathological disorders including cancer. During DNA metabolism, the primary causes of replication fork stalling include secondary DNA structures, highly transcribed regions and damaged DNA. The restart of stalled replication forks is critical for the timely progression of the cell cycle and ultimately for the maintenance of genomic stability. Our previous work has implicated the single-stranded DNA binding protein, hSSB1/NABP2, in the repair of DNA double-strand breaks via homologous recombination. Here, we demonstrate that hSSB1 relocates to hydroxyurea (HU)-damaged replication forks where it is required for ATR and Chk1 activation and recruitment of Mre11 and Rad51. Consequently, hSSB1-depleted cells fail to repair and restart stalled replication forks. hSSB1 deficiency causes accumulation of DNA strand breaks and results in chromosome aberrations observed in mitosis, ultimately resulting in hSSB1 being required for survival to HU and camptothecin. Overall, our findings demonstrate the importance of hSSB1 in maintaining and repairing DNA replication forks and for overall genomic stability.

Effect of soil properties on the response of pile to underground explosion

This paper develops and presents a fully coupled non-linear finite element procedure to treat the response of piles to ground shocks induced by underground explosions. The Arbitrary Lagrange Euler coupling formulation with proper state material parameters and equations are used in the study. Pile responses in four different soil types, viz, saturated soil, partially saturated soil and loose and dense dry soils are investigated and the results compared. Numerical results are validated by comparing with those from a standard design manual. Blast wave propagation in soils, horizontal pile deformations and damages in the pile are presented. The pile damage presented through plastic strain diagrams will enable the vulnerability assessment of the piles under the blast scenarios considered. The numerical results indicate that the blast performance of the piles embedded in saturated soil and loose dry soil are more severe than those in piles embedded in partially saturated soil and dense dry soil. Present findings should serve as a benchmark reference for future analysis and design.

Road infrastructure vulnerability to groundwater table variation due to sea level rise

This study was conducted to assess the vulnerability of coastal road infrastructures due to climate change induced sea level rise and extreme weather conditions through the estimation of road subgrade strength reduction as a result of changes in soil moisture content. The study area located in the Gold Coast, Australia highlighted that the risk is significant. In wet seasons or areas with wet condition, the groundwater table is already high, so even a small change in the groundwater table can raise the risk of inundation; particularly, in areas with existing shallow groundwater. The predicted risk of a high groundwater table on road infrastructure is a long-term hazard. Therefore, there is time to undertake some management plans to decrease the possible risks, for instance, some deep root plants could be planted along the roads with a high level of risk, to decrease the groundwater table elevation.