Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue, UV-A, and green light.

The Arabidopsis HY4 gene, required for blue-light-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a blue-light photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/far-red light or in the dark. This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from over-expression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal, transduction required by the CRY1 protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.

Hepatic expression of mature transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions.

Aberrant expression of transforming growth factor beta 1 (TGF-beta 1) has been implicated in a number of disease processes, particularly those involving fibrotic and inflammatory lesions. To determine the in vivo effects of overexpression of TGF-beta 1 on the function and structure of hepatic as well as extrahepatic tissues, transgenic mice were generated containing a fusion gene (Alb/TGF-beta 1) consisting of modified porcine TGF-beta 1 cDNA under the control of the regulatory elements of the mouse albumin gene. Five transgenic lines were developed, all of which expressed the Alb/TGF-beta 1 transgene selectively in hepatocytes. The transgenic line 25 expressing the highest level of the transgene in the liver also had high (> 10-fold over control) plasma levels of TGF-beta 1. Hepatic fibrosis and apoptotic death of hepatocytes developed in all the transgenic lines but was more pronounced in line 25. The fibrotic process was characterized by deposition of collagen around individual hepatocytes and within the space of Disse in a radiating linear pattern. Several extrahepatic lesions developed in line 25, including glomerulonephritis and renal failure, arteritis and myocarditis, as well as atrophic changes in pancreas and testis. The results from this transgenic model strongly support the proposed etiological role for TGF-beta 1 in a variety of fibrotic and inflammatory disorders. The transgenic model may also provide an appropriate paradigm for testing therapeutic interventions aimed at neutralizing the detrimental effects of this important cytokine.

Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression

Objectives: To identify potential molecular genetic determinants of cardiovascular ischemic tolerance in wild-type and transgenic hearts overexpressing A(1) adenosine receptors (A(1)ARs). Methods: cDNA microarrays were used to explore expression of 1824 genes ill wild-type hearts and ischemia-tolerant mouse hearts overexpressing A(1)ARs. Results: Overexpression of A(1)ARs reduced post-ischemic contractile dysfunction, limited arrhythmogenesis, and reduced necrosis by similar to80% in hearts subjected to 30 min global ischemia 60 mill reperfusion. Cardioprotection was abrogated by acute A(1)AR antagonism, and only a small number (19) of genes were modified by A(1)AR overexpression in normoxic hearts. Ischemia-reperfusion significantly altered expression of 75 genes in wild-type hearts (14 induced, 61 down-regulated), including genes for metabolic enzymes, structural/motility proteins, cell signaling proteins, defense/growth proteins, and regulators of transcription and translation. A(1)AR overexpression reversed the majority of gene down-regulation whereas gene induction was generally unaltered. Additionally, genes involved in cell defence, signaling and gene expression were selectively modified by ischemia in transgenic hearts (33 induced, 10 down-regulated), possibly contributing to the protected phenotype. Real-time PCR verified changes in nine selected genes, revealing concordance with array data. Transcription of the A(1)AR gene was also modestly reduced post-ischemia, consistent with impaired functional sensitivity to A(1)AR stimulation Conclusions: Data are presented regarding the early post-ischemic gene profile of intact heart. Reduced A(1)AR transcription is observed which may contribute to poor outcome from ischemia. A(1)AR overexpression selectively modifies post-ischemic gene expression, potentially contributing to ischemic-tolerance. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.

Overexpression of sonic hedgehog suppresses embryonic hair follicle morphogenesis

The Sonic Hedgehog (Shh) signalling pathway plays a central role in the development of the skin and hair follicle and is a major determinant of skin tumorigenesis, most notably of basal cell carcinoma (BCC). Various mouse models involving either ablation or overexpression of key members of the Shh signalling pathway display a range of skin tumours. To further examine the role of Shh in skin development. we have overexpressed Shh in a subset of interfollicular basal cells from 12.5 dpc under the control of the human keratin 1 (HK1) promoter. The HK1-Shh transgenic mice display a range of skin anomalies, including highly pigmented inguinal lesions and regions of alopecia. The most striking hair follicle phenotype is a suppression in embryonic follicle development between 14.0 and 19.0 dpc, resulting in a complete absence of guard, awl, and auchene hair fibres. These data indicate that alternative signals are responsible for the development of different hair follicles and point to a major role of Shh signalling in the morphogenesis of guard, awl, and auchene hair fibres. Through a comparison with other mouse models, the characteristics of the HK1-Shh transgenic mice suggest that the precise timing and site of Shh expression are key in dictating the resultant skin and tumour phenotype. 2003 Elsevier Inc. All rights reserved.

Overexpression of Leucyl Aminopeptidase in Plasmodium falciparum Parasites. Target for the antimalarial activity of bestatin

Malaria aminopeptidases are important in the generation and regulation of free amino acids that are used in protein anabolism and for maintaining osmotic stability within the infected erythrocyte. The intraerythrocytic development of malaria parasites is blocked when the activity of aminopeptidases is specifically inhibited by reagents such as bestatin. One of the major aminopeptidases of malaria parasites is a leucyl aminopeptidase of the M17 family. We reasoned that, when this enzyme was the target of bestatin inhibition, its overexpression in malaria cells would lead to a reduced sensitivity to the inhibitor. To address this supposition, transgenic Plasmodium falciparum parasites overexpressing the leucyl aminopeptidase were generated by transfection with a plasmid that housed the full-length gene. Transgenic parasites expressed a 65-kDa protein close to the predicted molecule size of 67.831 kDa for the introduced leucyl aminopeptidase, and immunofluorescence studies localized the protein to the cytosol, the location of the native enzyme. The product of the transgene was shown to be functionally active with cytosolic extracts of transgenic parasites exhibiting twice the leucyl aminopeptidase activity compared with wildtype parasites. In vitro inhibitor sensitivity assays demonstrated that the transgenic parasites were more resistant to bestatin (EC50 64 mu M) compared with the parent parasites (EC50 25 mu M). Overexpression of genes in malaria parasites would have general application in the identification and validation of targets for antimalarial drugs.

Overexpression Of Ucp1 In Tobacco Induces Mitochondrial Biogenesis And Amplifies A Broad Stress Response.

Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5'-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown. Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions. Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.

Evaluation Of Fe Uptake And Translocation In Transgenic And Non-transgenic Soybean Plants Using Enriched Stable (57)fe As A Tracer.

A tracer experiment is carried out with transgenic T (variety M 7211 RR) and non-transgenic NT (variety MSOY 8200) soybean plants to evaluate if genetic modification can influence the uptake and translocation of Fe. A chelate of EDTA with enriched stable (57)Fe is applied to the plants cultivated in vermiculite plus substrate and the (57)Fe acts as a tracer. The exposure of plants to enriched (57)Fe causes the dilution of the natural previously existing Fe in the plant compartments and then the changed Fe isotopic ratio ((57)Fe/(56)Fe) is measured using a quadrupole-based inductively coupled plasma mass spectrometer equipped with a dynamic reaction cell (DRC). Mathematical calculations based on the isotope dilution methodology allow distinguishing the natural abundance Fe from the enriched Fe (incorporated during the experiment). The NT soybean plants acquire higher amounts of Fe from natural abundance (originally present in the soil) and from enriched Fe (coming from the (57)Fe-EDTA during the experiment) than T soybean ones, demonstrating that the NT soybean plants probably absorb higher amounts of Fe, independently of the source. The percentage of newly incorporated Fe (coming from the treatment) was approximately 2.0 and 1.1% for NT and T soybean plants, respectively. A higher fraction (90.1%) of enriched Fe is translocated to upper parts, and a slightly lower fraction (3.8%) is accumulated in the stems by NT plants than by T ones (85.1%; 5.1%). Moreover, in both plants, the Fe-EDTA facilitates the transport and translocation of Fe to the leaves. The genetic modification is probably responsible for differences observed between T and NT soybean plants.

Sci1 Is A Component Of The Auxin-dependent Control Of Cell Proliferation In Arabidopsis Upper Pistil.

To characterize the recently described SCI1 (stigma/style cell cycle inhibitor 1) gene relationship with the auxin pathway, we have taken the advantage of the Arabidopsis model system and its available tools. At first, we have analyzed the At1g79200 T-DNA insertion mutants and constructed various transgenic plants. The loss- and gain-of-function plants displayed cell number alterations in upper pistils that were controlled by the amino-terminal domain of the protein. These data also confirmed that this locus holds the functional homolog (AtSCI1) of the Nicotiana tabacum SCI1 gene. Then, we have provided some evidences the auxin synthesis/signaling pathways are required for downstream proper AtSCI1 control of cell number: (a) its expression is downregulated in yuc2yuc6 and npy1 auxin-deficient mutants, (b) triple (yuc2yuc6sci1) and double (npy1sci1) mutants mimicked the auxin-deficient phenotypes, with no synergistic interactions, and (c) the increased upper pistil phenotype in these last mutants, which is a consequence of an increased cell number, was able to be complemented by AtSCI1 overexpression. Taken together, our data strongly suggests SCI1 as a component of the auxin signaling transduction pathway to control cell proliferation/differentiation in stigma/style, representing a molecular effector of this hormone on pistil development.

Prostatic Angiogenic Responses In Late Life: Antiangiogenic Therapy Influences And Relation With The Glandular Microenvironment In The Transgenic Adenocarcinoma Of Mouse Prostate (tramp) Model.

Aging is considered one of the main predisposing factors for the development of prostate malignancies. Angiogenesis is fundamental for tumor growth and its inhibition represents a promising therapeutic approach in cancer treatment. Thus, we sought to determine angiogenic responses and the effects of antiangiogenic therapy in the mouse prostate during late life, comparing these findings with the prostatic microenvironment in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model. Male mice (52 week-old FVB) were submitted to treatments with SU5416 (6 mg/kg; i.p.) and/or TNP-470 (15 mg/kg; s.c.). Finasteride was administered (20 mg/kg; s.c.), alone or in association to both inhibitors. The dorsolateral prostate was collected for VEGF, HIF-1α, FGF-2 and endostatin immunohistochemical and Western Blotting analyses and for microvessel density (MVD) count. Senescence led to increased MVD and VEGF, HIF-1α and FGF-2 protein levels in the prostatic microenvironment, similarly to what was observed in TRAMP mice prostate. The angiogenic process was impaired in all the treated groups, demonstrating significantly decreased MVD. Antiangiogenic and/or finasteride treatments resulted in decreased VEGF and HIF-1α levels, especially following TNP-470 administration, either alone or associated to SU5416. The combination of these agents resulted in increased endostatin levels, regardless of the presence of finasteride. Prostatic angiogenesis stimulation during senescence favored the development of neoplastic lesions, considering the pro-angiogenic microenvironment as a common aspect also observed during cancer progression in TRAMP mice. The combined antiangiogenic therapy was more efficient, leading to enhanced imbalance towards angiogenic inhibition in the organ. Finally, finasteride administration might secondarily upregulate the expression of pro-angiogenic factors, pointing to the harmful effects of this therapy. Prostate 75: 484-499, 2015. © 2014 Wiley Periodicals, Inc.

Comparative Study Of Transgenic And Non-transgenic Maize (zea Mays) Flours Commercialized In Brazil, Focussing On Proteomic Analyses.

Genetically modified foods are a major concern around the world due to the lack of information concerning their safety and health effects. This work evaluates differences, at the proteomic level, between two types of crop samples: transgenic (MON810 event with the Cry1Ab gene, which confers resistance to insects) and non-transgenic maize flour commercialized in Brazil. The 2-D DIGE technique revealed 99 differentially expressed spots, which were collected in 2-D PAGE gels and identified via mass spectrometry (nESI-QTOF MS/MS). The abundance of protein differences between the transgenic and non-transgenic samples could arise from genetic modification or as a result of an environmental influence pertaining to the commercial sample. The major functional category of proteins identified was related to disease/defense and, although differences were observed between samples, no toxins or allergenic proteins were found.

A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, ""promiscuous"" ( multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II-transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.

Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

Background: Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity. Results: Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system. Conclusions: Our results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

Ectopic expression of soybean leghemoglobin in chloroplasts impairs gibberellin biosynthesis and induces dwarfism in transgenic potato plants

We have characterized potato (Solanum tuberosum L.) plants expressing a soybean leghemoglobin that is targeted to plastids. Transgenic plants displayed a dwarf phenotype caused by short internode length, and exhibited increased tuberization in vitro. Under in vivo conditions that do not promote tuberization, plants showed smaller parenchymal cells than control plants. Analysis of gibberellin (GA) concentrations indicated that the transgenic plants have a substantial reduction (approximately 10-fold) of bioactive GA(1) concentration in shoots. Application of GA(3) to the shoot apex of the transformed plants completely restored the wild type phenotype suggesting that GA-biosynthesis rather than signal transduction was limiting. Since the first stage of the GA-biosynthetic pathway is located in the plastid, these results suggest that an early step in the pathway may be affected by the presence of the leghemoglobin.

Culturable endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane and its non-transgenic isolines

The diversity of endophytic filamentous fungi from leaves of transgenic imidazolinone-tolerant sugarcane plants and its isoline was evaluated by cultivation followed by amplified rDNA restriction analysis (ARDRA) of randomly selected strains. Transgenic and non-transgenic cultivars and their crop management (herbicide application or manual weed control) were used to assess the possible non-target effects of genetically modified sugarcane on the fungal endophytic community. A total of 14 ARDRA haplotypes were identified in the endophytic community of sugarcane. Internal transcribed spacer (ITS) sequencing revealed a rich community represented by 12 different families from the Ascomycota phylum. Some isolates had a high sequence similarity with genera that are common endophytes in tropical climates, such as Cladosporium, Epicoccum, Fusarium, Guignardia, Pestalotiopsis and Xylaria. Analysis of molecular variance indicated that fluctuations in fungal population were related to both transgenic plants and herbicide application. While herbicide applications quickly induced transient changes in the fungal community, transgenic plants induced slower changes that were maintained over time. These results represent the first draft on composition of endophytic filamentous fungi associated with sugarcane plants. They are an important step in understanding the possible effects of transgenic plants and their crop management on the fungal endophytic community.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.