A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

An egg apparatus-specific enhancer of Arabidopsis, identified by enhancer detection

Despite a central role in angiosperm reproduction, few gametophyte-specific genes and promoters have been isolated, particularly for the inaccessible female gametophyte (embryo sac). Using the Ds-based enhancer-detector line ET253, we have cloned an egg apparatus-specific enhancer (EASE) from Arabidopsis (Arabidopsis thaliana). The genomic region flanking the Ds insertion site was further analyzed by examining its capability to control gusA and GFP reporter gene expression in the embryo sac in a transgenic context. Through analysis of a 5' and 3' deletion series in transgenic Arabidopsis, the sequence responsible for egg apparatus-specific expression was delineated to 77 bp. Our data showed that this enhancer is unique in the Arabidopsis genome, is conserved among different accessions, and shows an unusual pattern of sequence variation. This EASE works independently of position and orientation in Arabidopsis but is probably not associated with any nearby gene, suggesting either that it acts over a large distance or that a cryptic element was detected. Embryo-specific ablation in Arabidopsis was achieved by transactivation of a diphtheria toxin gene under the control of the EASE. The potential application of the EASE element and similar control elements as part of an open-source biotechnology toolkit for apomixis is discussed.

In vivo knockdown of the androgen receptor results in growth inhibition and regression of well-established, castration-resistant prostate tumours

Purpose: Progression to the castration-resistant state is the incurable and lethal end stage of prostate cancer, and there is strong evidence that androgen receptor (AR) still plays a central role in this process. We hypothesize that knocking down AR will have a major effect on inhibiting growth of castration-resistant tumors. Experimental Design: Castration-resistant C4-2 human prostate cancer cells stably expressing a tetracycline-inducible AR-targeted short hairpin RNA (shRNA) were generated to directly test the effects of AR knockdown in C4-2 human prostate cancer cells and tumors. Results:In vitro expression of AR shRNA resulted in decreased levels of AR mRNA and protein, decreased expression of prostate-specific antigen (PSA), reduced activation of the PSA-luciferase reporter, and growth inhibition of C4-2 cells. Gene microarray analyses revealed that AR knockdown under hormone-deprived conditions resulted in activation of genes involved in apoptosis, cell cycle regulation, protein synthesis, and tumorigenesis. To ensure that tumors were truly castration-resistant in vivo, inducible AR shRNA expressing C4-2 tumors were grown in castrated mice to an average volume of 450 mm3. In all of the animals, serum PSA decreased, and in 50% of them, there was complete tumor regression and disappearance of serum PSA. Conclusions: Whereas castration is ineffective in castration-resistant prostate tumors, knockdown of AR can decrease serum PSA, inhibit tumor growth, and frequently cause tumor regression. This study is the first direct evidence that knockdown of AR is a viable therapeutic strategy for treatment of prostate tumors that have already progressed to the castration-resistant state.

Disruption of androgen regulation in the prostate by the environmental contaminant hexachlorobenzene

Hexachlorobenzene (HCB) is a persistent environmental contaminant that has the potential to interfere with steroid hormone regulation. The prostate requires precise control by androgens to regulate its growth and function. To determine if HCB impacts androgen action in the prostate, we used a number of methods. Our in vitro cell-culture-based assay used a firefly luciferase reporter gene driven by an androgen-responsive promoter. In the presence of dihydrotestosterone, low concentrations (0.5-5 nM) of HCB increased the androgen-responsive production of firefly luciferase and high concentrations of HCB (> 10 microM) suppressed this transcriptional activity. Results from a binding assay showed no evidence of affinity between HCB and the androgen receptor. We also tested HCB for in vivo effects using transgenic mice in which the transgene was a prostate-specific, androgen-responsive promoter upstream of a chloramphenicol acetyl transferase (CAT) reporter gene. In 4-week-old mice, the proportion of dilated prostate acini, a marker of sexual maturity, increased in the low HCB dose group and decreased in the high HCB dose mice. In the 8-week-old mice, there was a significant decrease in both CAT activity and prostate weight upon exposure to 20 mg/kg/day HCB. Therefore, in vitro and in vivo data suggest that HCB weakly agonizes androgen action, and consequently, low levels of HCB enhanced androgen action but high levels of HCB interfered. Environmental contaminants have been implicated in the rising incidence of prostate cancer, and insight into the mechanisms of endocrine disruption will help to clarify their role.

The relationship of boat movements to environmental management at some Queensland marinas

Marinas currently exist primarily to service recreational boats, and these vessels are a potential cause of both problems and opportunities in environmental management. Thus, on the one hand, destructive fuel and other pollutants may be expelled, boat wakes can cause littoral soil erosion, physical damage results from collisions with marine life, and litter and noise pollution occur in otherwise pristine habitat. Boats also provide access to otherwise inaccessible natural environments for educational and other management reasons. In this study, boat traffic at three large marinas located along the Queensland coastline has been field surveyed for introductory information. No attempt was made at this juncture to survey the behaviour of the boat crews and passengers (concerning actual destinations, activities on board, etc. or to survey the recreational boat industry. Such studies rely on boat registration records and personal questionnaires. Some other surveys relating to fishing draw on boat ramp surveys and direct submissions by recreational fishers; these provide some data on daily usage of boat ramps, but without particular attention to boats. We believe field observations of overall boat activities in the water are necessary for environmental management purposes. The aim of the survey was to provide information to help prioritize the potential impacts that boats’ activities have on the surrounding natural environment. Any impact by boats will be a product of their numbers, size, frequency of movement, carrying capacity and routes/destinations. The severity of impacts will dictate the appropriate management action.

Methods to achieve net environmental gain in the course of development in Queensland

To achieve best environmental management practice in Queensland, effort needs to be extended into the private sector. A Regional Landscape Strategy compiled for any substantial new proposal must identify the most promising technique(s) (from an available tool kit of 13) by which a developer (of any type) is more likely to sustain on-site resources while assisting government deliver its future plans in any region of the State. Offsetting may prove to be one of the most effective of these tools. However, policy must address‘offset land mitigation’, whereby the necessary financial incentives are introduced. Practicable methods by which offset sites can be selected, and measurement of their consequent environmental benefit, have now been devised and tested to assist this process.

A site selection process for environmental offsetting purposes in Queensland

A method of selecting land in any region of Queensland for offsetting purposes is devised, employing uniform standards. The procedure first requires that any core natural asset lands, Crown environmental lands, prime urban and agricultural lands, and highly contentious sites in the region be eliminated from consideration. Other land is then sought that is located between existing large reservations and the centre of greatest potential regional development/disturbance. Using the criteria of rehabilitation (rather than preservation) plus proximity to those officially defined Regional Ecosystems that are most threatened, adjacent sites that are described as ‘Cleared’ are identified in terms of agricultural land capability. Class IV lands – defined as those ‘which may be safely used for occasional cultivation with careful management’,2 ‘where it is favourably located for special usage’,3 and where it is ‘helpful to those who are interested in industry or regional planning or in reconstruction’4 – are examined for their appropriate area, for current tenure and for any conditions such as Mining Leases that may exist. The positive impacts from offsets on adjoining lands can then be designed to be significant; examples are also offered in respect of riparian areas and of Marine Parks. Criteria against which to measure performance for trading purposes include functional lift, with other case studies about this matter reported separately in this issue. The procedure takes no account of demand side economics (financial additionality), which requires commercial rather than environmental analysis.

Environmental management and wildlife in Queensland : a review of Queensland mammal species designated as scarce

From 19 authoritative lists with 164 entries of ‘endangered’ Australian mammal species, 39 species have been reported as extinct. When examined in the light of field conditions, the 18 of these species thought to be from Queensland consist of (a) species described from fragmentary museum material collected in the earliest days of exploration, (b) populations inferred to exist in Queensland by extrapolation from distribution records in neighbouring States or countries, (c) inhabitants of remote and harsh locations where search effort is extraordinarily difficult (especially in circumstances of drought or flooding). and/or (d) individuals that are clearly transitory or peripheral in distribution. ‘Rediscovery’ of such scarce species - a not infrequent occurrence - is nowadays attracting increasing attention. Management in respect of any scarce wildlife in Queensland presently derives from such official lists. The analyses here indicate that this method of prioritizing action needs review. This is especially so because action then tends to be centred on species chosen out of the lists for populist reasons and that mostly addresses Crown lands. There is reason to believe that the preferred management may lie private lands where casual observation has provided for rediscovery and where management is most desirable and practicable.

Measurement of the net environmental benefit of offsetting in Queensland

The process of offsetting land against unavoidable disturbance of development sites in Queensland will benefit from a method that allows the best possible selection to be made of alternative lands. With site selection now advocated through a combination of Regional Ecosystem and Land Capability classifications state-wide, a case study has determined methods of assessing the functional lift – that is, measures of net environmental gain – of such action. Outcomes with potentially high functional lift are determined, that offer promise not only for endangered ecosystems but also for managing adjacent conservation reserves.

Management of a tributyl tin issue in a marina development in Queensland

Tributyl tin (TBT) deposits in the sediments are one of many impacts that have been imposed on both the environment and the up-coming development of Boat Haven, Airlie Beach, Queensland. The current costly solution to this problem (that is, removal and re-burial) could be put in future to the credit of the developer rather than be treated (as at present) as a penalty. The Queensland Government’s Offsets Scheme provides an opportunity to promote effective conservation of regional landscapes. Because this scheme plans for offsetting in terrestrial vegetation systems through rehabilitation, so credits could be given to those approved developers who rehabilitate valuable marine habitats disturbed by TBT deposits.

Elements of an environmental offsets policy : a working paper towards a policy for an environmental banking scheme appropriate for Queensland

In the past eight years, Australia has adopted the use of environmental offsets as a means to compensate for environmental degradation from development. Queensland has more environmental offsetting policies than any other Australian State or Territory. The methodology has profound effects on development companies, landowners (both private and public), regional land planning, organizations, government agencies, monetary banking institutions and environmental conservation bodies.

A novel corepressor, BCoR-L1, represses transcription through an interaction with CtBP

Corepressors play a crucial role in negative gene regulation and are defective in several diseases. BCoR is a corepressor for the BCL6 repressor protein. Here we describe and functionally characterize BCoR-L1, a homolog of BCoR. When tethered to a heterologous promoter, BCoR-L1 is capable of strong repression. Like other corepressors, BCoR-L1 associates with histone deacetylase (HDAC) activity. Specifically, BCoR-L1 coprecipitates with the Class II HDACs, HDAC4, HDAC5, and HDAC7, suggesting that they are involved in its role as a transcriptional repressor. BCoR-L1 also interacts with the CtBP corepressor through a CtBP-interacting motif in its amino terminus. Abrogation of the CtBP binding site within BCoR-L1 partially relieves BCoR-L1-mediated transcriptional repression. Furthermore, BCoR-L1 is located on the E-cadherin promoter, a known CtBP-regulated promoter, and represses the E-cadherin promoter activity in a reporter assay. The inhibition of BCoR-L1 expression by RNA-mediated interference results in derepression of E-cadherin in cells that do not normally express E-cadherin, indicating that BCoR-L1 contributes to the repression of an authentic endogenous CtBP target.

A variant of the KLK4 gene is expressed as a cis sense-antisense chimeric transcript in prostate cancer cells

In humans, more than 30,000 chimeric transcripts originating from 23,686 genes have been identified. The mechanisms and association of chimeric transcripts arising from chromosomal rearrangements with cancer are well established, but much remains unknown regarding the biogenesis and importance of other chimeric transcripts that arise from nongenomic alterations. Recently, a SLC45A3–ELK4 chimera has been shown to be androgen-regulated, and is overexpressed in metastatic or high-grade prostate tumors relative to local prostate cancers. Here, we characterize the expression of a KLK4 cis sense–antisense chimeric transcript, and show other examples in prostate cancer. Using non-protein-coding microarray analyses, we initially identified an androgen-regulated antisense transcript within the 3′ untranslated region of the KLK4 gene in LNCaP cells. The KLK4 cis-NAT was validated by strand-specific linker-mediated RT-PCR and Northern blotting. Characterization of the KLK4 cis-NAT by 5′ and 3′ rapid amplification of cDNA ends (RACE) revealed that this transcript forms multiple fusions with the KLK4 sense transcript. Lack of KLK4 antisense promoter activity using reporter assays suggests that these transcripts are unlikely to arise from a trans-splicing mechanism. 5′ RACE and analyses of deep sequencing data from LNCaP cells treated ±androgens revealed six high-confidence sense–antisense chimeras of which three were supported by the cDNA databases. In this study, we have shown complex gene expression at the KLK4 locus that might be a hallmark of cis sense–antisense chimeric transcription.

Journalists at war

Journalism is an especially hazardous profession when it takes the reporter into zones of war and conflict. The Committee to Protect Journalists records that in 2010 44 journalists were killed while carrying out their duties. Some of these were reporting conflict in Afghanistan, Somalia, Iraq and elsewhere. Others were on assignments covering crime and corruption in Mexico, Russia, Venezuela*all places where telling truth to power can easily get you killed, beaten or banged up. In the last 20 years some 874 journalists have been killed on the job, and we salute them all. Journalists get criticised a lot by we scholars, and often for good reason. They can be villains, for sure, but they can also be heroes, when they lay down their lives in the pursuit of the truth. As this piece was being edited, photojournalists Tim Hetherington and Chris Hondros were killed in Libya.

Multiple analysis of three common genetic alterations associated with thrombophilia

We have previously reported the use of a novel mini-sequencing protocol for detection of the factor V Leiden variant, the first nucleotide change (FNC) technology. This technology is based on a single nucleotide extension of a primer, which is hybridized immediately adjacent to the site of mutation. The extended nucleotide that carries a reporter molecule (fluorescein) has the power to discriminate the genotype at the site of mutation. More recently, the prothrombin 20210 and thermolabile methylene tetrahydrofolate reductase (MTHFR) 677 variants have been identified as possible risk factors associated with thrombophilia. This study describes the use of the FNC technology in a combined assay to detect factor V, prothrombin and MTHFR variants in a population of Australian blood donors, and describes the objective numerical methodology used to determine genotype cut-off values for each genetic variation. Using FNC to test 500 normal blood donors, the incidence of Factor V Leiden was 3.6% (all heterozygous), that of prothrombin 20210 was 2.8% (all heterozygous) and that of MTHFR was 10% (homozygous). The combined FNC technology offers a simple, rapid, automatable DNA-based test for the detection of these three important mutations that are associated with familial thrombophilia. (C) 2000 Lippincott Williams and Wilkins.