Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants

A series of improved vectors have been constructed that are suitable for use in Agrobacterium tumefaciens-mediated monocot transformation. These binary vectors have several useful features, including the selectable marker genes bar (phosphinothricin resistance) or hph (hygromycin resistance) driven by either the cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin promoter, a high-copy-number replication origin that allows reliable mini-prep DNA isolation from Escherichia coli, and a polylinker sequence into which target genes can be easily inserted. A significant improvement has been made to the hph gene by the introduction of an intron into its coding region. The presence of the intron abolishes hph expression in A. tumefaciens, rendering the bacterium susceptible to the selective agent hygromycin B. The use of such an intron-hph vector thus enables the antibiotic in plant culture media to function as both a selective agent for transformed tissue and as a contraselective agent for A. tumefaciens growth, thus minimising the overgrowth of A. tumefaciens on plant tissues during transformation. Furthermore, the intron appears to be correctly spliced in plant cells and significantly enhances hph expression in transformed rice tissue. In our experiments, the use of the intron-hph vector increased the frequency of rice transformation and has enabled the production of transgenic barley.

Jane McCredie - Making Girls and Boys : Inside the Science of Sex [Book Review]

Activists, Feminists, queer theorists, and those who live outside traditional gender narratives have long challenged the fixity of the sex and gender binaries. While the dominant Western paradigm posits sex and gender as natural and inherent, queer theory argues that sex and gender are socially constructed. This means that our ideas about sex and gender, and the concepts themselves, are shaped by particular social contexts. Questioning the nature of sex can be puzzling. After all, isn’t sex biology? Binary sex – male and female – was labelled as such by scientists based on existing binary categories and observations of hormones, genes, chromosomes, reproductive organs, genitals and other bodily elements. Binary sex is allocated at birth by genital appearance. Not everyone fits into these categories and this leads queer theorists, and others, to question the categories. Now, “some scientists are also starting to move away from the idea of biology as the fixed basis on which the social artefact of gender is built” (5). Making Girls and Boys: Inside the Science of Sex, by Jane McCredie, examines theories about gender roles and behaviours also considering those who don’t fit the arbitrary sex and gender binaries.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d/r) or inverted-repeat (i/r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i/r T-DNA insertions is a primary inducer of transgene silencing in plants. © CSIRO 2005.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successful Agrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph (encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4-fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 carrying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher β-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity comparable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens

In binary vectors, the antibiotic resistance gene used for selection of transformed plant cells is also usually expressed in the transforming Agrobacterium cells. This expression gives the bacterium antibiotic resistance, an unnecessary advantage on selective medium containing the antibiotic. Insertion of a castor bean catalase-1 (CAT-1) gene intron or a Parasponia andersonii haemoglobin gene intron into the coding region of the selectable marker gene, hph, completely abolished the expression of the gene in Agrobacterium, rendering it susceptible to hygromycin B. Use of these modified binary vectors minimized the overgrowth of Agrobacterium during plant transformation. Both of the introns were correctly spliced in plant cells and significantly enhanced hph gene expression in transformed rice tissue. The presence of these introns in the hph coding sequence not only maintained the selection efficiency of the hph gene, but with the CAT-1 intron also substantially increased the frequency of rice transformation. Transgenic lines with an intron-hph gene generally contained fewer gene copies and produced substantially more mRNA of the predicted size. Our results also indicate that transgenic plants with many copies of the transgene were more likely to show gene silencing than plants with 1-3 copies.

Quality based frame selection for face clustering in news video

Clustering identities in a broadcast video is a useful task to aid in video annotation and retrieval. Quality based frame selection is a crucial task in video face clustering, to both improve the clustering performance and reduce the computational cost. We present a frame work that selects the highest quality frames available in a video to cluster the face. This frame selection technique is based on low level and high level features (face symmetry, sharpness, contrast and brightness) to select the highest quality facial images available in a face sequence for clustering. We also consider the temporal distribution of the faces to ensure that selected faces are taken at times distributed throughout the sequence. Normalized feature scores are fused and frames with high quality scores are used in a Local Gabor Binary Pattern Histogram Sequence based face clustering system. We present a news video database to evaluate the clustering system performance. Experiments on the newly created news database show that the proposed method selects the best quality face images in the video sequence, resulting in improved clustering performance.

Optimal camera planning under versatile user constraints in multi-camera image processing systems

The selection of optimal camera configurations (camera locations, orientations, etc.) for multi-camera networks remains an unsolved problem. Previous approaches largely focus on proposing various objective functions to achieve different tasks. Most of them, however, do not generalize well to large scale networks. To tackle this, we propose a statistical framework of the problem as well as propose a trans-dimensional simulated annealing algorithm to effectively deal with it. We compare our approach with a state-of-the-art method based on binary integer programming (BIP) and show that our approach offers similar performance on small scale problems. However, we also demonstrate the capability of our approach in dealing with large scale problems and show that our approach produces better results than two alternative heuristics designed to deal with the scalability issue of BIP. Last, we show the versatility of our approach using a number of specific scenarios.

Transcutaneous immunization with combined cholera toxin and CpG adjuvant protects against Chlamydia muridarum genital tract infection

Chlamydia trachomatis is a pathogen of the genital tract and ocular epithelium. Infection is established by the binding of the metabolically inert elementary body (EB) to epithelial cells. These are taken up by endocytosis into a membrane-bound vesicle termed an inclusion. The inclusion avoids fusion with host lysosomes, and the EBs differentiate into the metabolically active reticulate body (RB), which replicates by binary fission within the protected environment of the inclusion. During the extracellular EB stage of the C. trachomatis life cycle, antibody present in genital tract or ocular secretions can inhibit infection both in vivo and in tissue culture. The RB, residing within the intracellular inclusion, is not accessible to antibody, and resolution of infection at this stage requires a cell-mediated immune response mediated by gamma interferon-secreting Th1 cells. Thus, an ideal vaccine to protect against C. trachomatis genital tract infection should induce both antibody (immunoglobulin A [IgA] and IgG) responses in mucosal secretions to prevent infection by chlamydial EB and a strong Th1 response to limit ascending infection to the uterus and fallopian tubes. In the present study we show that transcutaneous immunization with major outer membrane protein (MOMP) in combination with both cholera toxin and CpG oligodeoxynucleotides elicits MOMP-specific IgG and IgA in vaginal and uterine lavage fluid, MOMP-specific IgG in serum, and gamma interferon-secreting T cells in reproductive tract-draining caudal and lumbar lymph nodes. This immunization protocol resulted in enhanced clearance of C. muridarum (C. trachomatis, mouse pneumonitis strain) following intravaginal challenge of BALB/c mice.

Efficient top-k retrieval with signatures

This paper describes a new method of indexing and searching large binary signature collections to efficiently find similar signatures, addressing the scalability problem in signature search. Signatures offer efficient computation with acceptable measure of similarity in numerous applications. However, performing a complete search with a given search argument (a signature) requires a Hamming distance calculation against every signature in the collection. This quickly becomes excessive when dealing with large collections, presenting issues of scalability that limit their applicability. Our method efficiently finds similar signatures in very large collections, trading memory use and precision for greatly improved search speed. Experimental results demonstrate that our approach is capable of finding a set of nearest signatures to a given search argument with a high degree of speed and fidelity.

Physical activity patterns and correlates among adults from a developing country : the Sri Lanka Diabetes and Cardiovascular Study

OBJECTIVE: To evaluate patterns of physical activity (PA), the prevalence of physical inactivity and the relationships between PA and sociodemographic, clinical and biochemical parameters among Sri Lankan adults. DESIGN: Descriptive cross-sectional study. SETTING: Nationally representative population-based survey conducted in Sri Lanka. SUBJECTS: Data on PA and associated details were obtained from 5000 adults. PA was assessed using the International Physical Activity Questionnaire (short-form). A binary logistic regression analysis was performed using the dichotomous variable ‘health-enhancing PA’ (05‘active’, 15‘inactive’). RESULTS: Sample size was 4485. Mean age was 46.1 (SD 15.1) years, 39.5% were males. The mean weekly total MET (metabolic equivalents of task) minutes of PA among the study population was 4703 (SD 4369). Males (5464 (SD 5452)) had a significantly higher weekly total MET minutes than females (4205 (SD 3394); P,0.001). Rural adults (5175 (SD 4583)) were significantly more active than urban adults (2956 (SD 2847); P<0.001). Tamils had the highest mean weekly total MET minutes among ethnicities. Those with tertiary education had lowest mean weekly total MET minutes. In all adults 60.0% were in the ‘highly active’ category, while only 11.0% were ‘inactive’ (males 14.6%, females 8.7%; P<0.001). Of the ‘highly active’ adults, 85.8% were residing in rural areas. Results of the binary logistic regression analysis indicated that female gender (OR52?1), age .70 years (OR53.8), urban living (OR52.5), Muslim ethnicity (OR52.7), tertiary education (OR53.6), obesity (OR51.8), diabetes (OR51.6), hypertension (OR51.2) and metabolic syndrome (OR51.3) were all associated with significantly increased odds of being physically ‘inactive’. CONCLUSIONS: The majority of Sri Lankan adults were ‘highly active’ physically. Female gender, older age, urban living, Muslim ethnicity and tertiary education were all significant predictors of physical inactivity. Physical inactivity was associated with obesity, diabetes, hypertension and metabolic syndrome.

Radiative and total heat transfer measurements to a Titan Explorer model

Radiative and total heat transfer at the flow stagnation point of a 1:40.8 binary scaled model of the Titan Explorer vehicle were measured in the X3 expansion tube. Results from the current study illustrated that with the addition of CH4 into a N2 test gas radiative heat transfer could be detected. For a test gas of 5% CH4 and 95% N2, simulating an atmospheric model for Titanic aerocapture, approximately 4% of the experimentally measured total stagnation point heat transfer was found to be due to radiation. This was in comparison to < 1% measured for a test gas of pure nitrogen. When scaled to the flight vehicle, experimental results indicate a 64% contribution of radiation (test gas 5% CH4/95% N2). Previous numerical results however have predicted this contribution to be between 80-92%. Thus, experimental results from the current study suggest that numerical analyses are over-predicting the radiative heat transfer on the flight vehicle.

Heat transfer measurements on the first experimental layer of the FIRE II reentry vehicle in expansion tubes

The present study focused on simulating a trajectory point towards the end of the first experimental heatshield of the FIRE II vehicle, at a total flight time of 1639.53s. Scale replicas were sized according to binary scaling and instrumented with thermocouples for testing in the X1 expansion tube, located at The University of Queensland. Correlation of flight to experimental data was achieved through the separation, and independent treatment of the heat modes. Preliminary investigation indicates that the absolute value of radiant surface flux is conserved between two binary scaled models, whereas convective heat transfer increases with the length scale. This difference in the scaling techniques result in the overall contribution of radiative heat transfer diminishing to less than 1% in expansion tubes from a flight value of approximately 9-17%. From empirical correlation's it has been shown that the St √Re number decreases, under special circumstances, in expansion tubes by the percentage radiation present on the flight vehicle. Results obtained in this study give a strong indication that the relative radiative heat transfer contribution in the expansion tube tests is less than that in flight, supporting the analysis that the absolute value remains constant with binary scaling.

The role of spreadsheets in an investigation of Fibonacci numbers

We introduce a function Z(k) which measures the number of distinct ways in which a number can be expressed as the sum of Fibonacci numbers. Using a binary table and other devices, we explore the values that Z(k) can take and reveal a surprising relationship between the values of Z(k) and the Fibonacci numbers from which they were derived. The article shows the way in which standard spreadsheet functionalities makes it possible to reveal quite striking patterns in data.

A new plasmid display technology for the in vitro selection of functional phenotype–genotype linked proteins

Background: Display technologies which allow peptides or proteins to be physically associated with the encoding DNA are central to procedures which involve screening of protein libraries in vitro for new or altered function. Here we describe a new system designed specifically for the display of libraries of diverse, functional proteins which utilises the DNA binding protein nuclear factor κB (NF-κB) p50 to establish a phenotype–genotype link between the displayed protein and the encoding gene. Results: A range of model fusion proteins to either the amino- or carboxy-terminus of NF-κB p50 have been constructed and shown to retain the picomolar affinity and DNA specificity of wild-type NF-κB p50. Through use of an optimal combination of binding buffer and DNA target sequence, the half-life of p50–DNA complexes could be increased to over 47 h, enabling the competitive selection of a variety of protein–plasmid complexes with enrichment factors of up to 6000-fold per round. The p50-based plasmid display system was used to enrich a maltose binding protein complex to homogeneity in only three rounds from a binary mixture with a starting ratio of 1:108 and to enrich to near homogeneity a single functional protein from a phenotype–genotype linked Escherichia coli genomic library using in vitro functional selections. Conclusions: A new display technology is described which addresses the challenge of functional protein display. The results demonstrate that plasmid display is sufficiently sensitive to select a functional protein from large libraries and that it therefore represents a useful addition to the repertoire of display technologies.