Combination of viral promoter sequences to generate highly active promoters for heterologous therapeutic protein over-expression in plants

To develop strong promoters for protein over-expression in both dicots and monocots, we constructed a new family of chimeric promoters using sequences of the Commelina Yellow Mosaic Virus (CoYMV), of the Cassava Vein Mosaic Virus (CsVMV) and activating sequences from the CaMV 35S promoter. The chimeric promoters were cloned upstream from the gusA reporter gene. The constructs were used in transient expression experiments, via DNA-coated gold particle delivery to tobacco leaves and maize endosperms. The results showed that candidates among the chimeric promoters could drive expression of the reporter gene to very high levels in the dicot plant tobacco, and all chimeric promoters showed higher expression in maize endosperm than the maize γ-zein promoter used as reference for the monocot expression. Expression cassettes were then used in stable tobacco transformation. Determination of GUS activity throughout growth of the primary transformants showed that two promoters (MPr1163 and MPr1165) could drive expression three to five-fold higher than the highly efficient enhanced 35S promoter. The use of MPr1163 was additionally validated for successful heterologous protein production of human lactoferrin in tobacco via agroinfiltration.     

Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures

Arabidopsis thaliana has emerged as a model species for the analysis of genes controlling plant development. However, its small size has impaired biochemical analyses, and the absence of a transient expression system has hampered promoter analysis. Here, we report a method for rapidly establishing A. thaliana suspension cultures that yield protoplasts that can be readily transfected. We have optimized transient expression conditions using a modified polyethylene glycol / calcium nitrate transformation protocol and a Cauliflower Mosaic Virus 35S promoter-luciferase reporter gene construct. Our methods permit isolation of large quantities of rapidly growing cells and analysis of Arabidopsis promoters in vivo in a homologous system.Abbreviations CaMV Cauliflower Mosaic Virus - 2,4D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)ethanesulfonic acid - PEG polyethylene glycol     

两株海洋真菌的鉴定及其次级代谢产物抑制烟草花叶病毒及抗肿瘤活性

摘要：【目的】通过对2株活性海洋真菌发酵产物提取物抑制烟草花叶病毒和抗肿瘤活性进行研究,为进一步得到活性纯品化合物作为抗病毒及抗肿瘤的先导化合物奠定基础。【方法】菌株发酵产物的粗提物是通过甲醇浸取并在真空条件下蒸干得到的。粗提物中溶于水的部分为水溶性部分,不溶于水的部分为脂溶性部分。通过间接酶联免疫法检测样品抑制烟草花叶病毒的活性,通过四甲基偶氮唑盐微量酶反应比色法（MTT法）检测样品抗肿瘤活性,通过形态及ITS rDNA序列法进行菌株鉴定。【结果】两株海洋真菌抑制烟草花叶病毒活性和抗肿瘤的活性均较高。分子鉴定结果显示,两株真菌分别与Penicillium oxalicum 和 Neosartorya fischeri 的同源性极高。菌株0312F1发酵液的水溶性部分具有抗病毒及抗肿瘤活性,菌株1008F1发酵液的脂溶性部分具有抑制烟草花叶病毒活性,而水溶性部分具有抗肿瘤活性。【结论】菌株0312F1和菌株1008F1发酵液的提取物抑制烟草花叶病毒的活性部位不同,而抗肿瘤活性部位相同。菌株0312F1发酵液提取物的水溶性活性部位对肝癌细胞BEL-7404的抑制效果比对胃癌细胞SGC-7901的抑制效果明显,而菌株1008F1发酵液提取物的水溶性活性部位对胃癌细胞SGC-7901的抑制效果比对肝癌细胞BEL-7404的抑制效果明显。     

Synthesis of tobacco mosaic virus coat protein following migration of viral RNA into isolated mouse liver mitochondria.

RNA of Tobacco Mosaic Virus is shown to be able to migrate into isolated mouse liver mitochondria, whence it can be reisolated intact. The migration of RNA is accompanied by enhanced rate of protein synthesis, which is sensitive to chloramphenicol, but not to cycloheximide. Evidence is presented showing that, among the products formed is the coat protein of Tobacco Mosaic Virus.     

In vitro synthesis of turnip yellow mosaic virus coat protein in a wheat germ cell-free system

Turnip Yellow Mosaic Virus RNA directs the synthesis in vitro of its coat protein in a wheat germ cell-free extract. Optimum conditions for synthesis have been defined, and the effect of spermine on specifically enhancing coat protein formation has been examined. Identity between the in vitro synthesized coat protein and authentic coat protein of Turnip Yellow Mosaic Virus was established by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunoprecipitation.     

The Effects of B, K10, and AR Chromosomes on the Resistance of Maize to Viral Infection

Studies were conducted to determine if accessory (B) chromosomes, the abnormal tenth (K10) chromosome or the aberrant ratio (AR) phenomenon of maize (Zea mays L.) affect the resistance of the plants to viral infection. Genetically similar stocks of maize with and without these elements were compared to determine what effect they would have on the plants response to Brome Mosaic Virus (BMV), Maize Dwarf Mosaic Virus (MDMV), Wheat Streak Mosaic Virus (WSMV) and Barley Stripe Mosaic Virus (BSMV).—The test results with BSMV were not found to be conclusive. With BMV and MDMV, neither the B orK10 chromosomes were found to alter infections; however, these chromosomes were found to affect the resistance of the plants to WSMV infection. The B chromosomes were found to delay the onset of leaf necrosis by 15%, while the K10 chromosome was found to increase the susceptibility to necrosis by 100%. The AR phenomenon was not found to alter the resistance of maize to BMV infection. However, it was found to increase the susceptibility of maize to MDMV infection by 36% and to decrease the susceptibility of maize to WSMV infection by 92%.     

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV Plum Pox Virus - CP coat protein - CaMV Cauliflower Mosaic Virus - CP+ coat protein expressing plant - CP– control plant = non coat protein expressing plant - TMV Tobacco Mosaic Virus - NPTII neomycin phosphotransferaseII - IBA indole-3-butyric acid - BAP 6-benzylaminopurine; - MS Murashige Skoog - ELISA enzyme linked immunosorbent assay     

Elastic properties of viruses

Viruses are compact biological nanoparticles whose elastic and dynamical properties are hardly known. Inelastic (Brillouin) light scattering was used to characterize these properties, from microcrystals of the Satellite Tobacco Mosaic Virus, a nearly spherical plant virus of 17-nm diameter. Longitudinal sound velocities in wet and dry Satellite Tobacco Mosaic Virus crystals were determined and compared to that of the well-known protein crystal, lysozyme. Localized vibrational modes of the viral particles (i.e., particle modes) were sought in the relevant frequency ranges, as derived assuming the viruses as full free nanospheres. Despite very favorable conditions, regarding virus concentration and expected low damping in dry microcrystals, no firm evidence of virus particle modes could be detected.     

Biological and biophysical properties of Haemophilus influenzae transforming DNA encapsided by viral proteins

Summary The expression of the transforming ability of Haemophilus influenzae DNA was investigated after its encapsidation by the coat protein of two different plant viruses, Brome Mosaic Virus (BMV) and Alfalfa Mosaic Virus (AMV). The influence of the encapsidation on the various steps of the transformation process was studied, as well as the protection of the DNA molecule inside of the DNA-protein complex particles against nucleolytic attack.The kinetics of uptake and penetration of free and encapsided DNA and their respective competitive abilities were compared in order to explain the differences which appeared between the rates of transformation by free and encapsided high molecular weight DNAs.Finally, some conclusions are drawn concerning the uncoating process of these nucleoprotein complex particles and the strength of the DNA-protein and protein-protein interactions existing in these particles.Abbreviations M.W. molecular weight - T.A. transforming ability     

Molecular mapping and genetic fine-structure of the rym5 locus encoding resistance to different strains of the Barley Yellow Mosaic Virus Complex

 The genetic structure of the rym5 locus was studied in a population comprising 391 doubled-haploid lines that were evaluated for resistance to two strains of Barley Yellow Mosaic Virus (BaYMV-1, 2) and to Barley Mild Mosaic Virus (BaMMV). The absence of recombinants that are able to differentiate between the reaction to these different bymoviruses provides evidence that rym5 is a complex locus, which is either composed of several closely linked genes or of an allelic series of a single gene. For marker-assisted introgression of this locus into adapted barley germplasm, a CAPS (cleaved amplified polymorphic sequence) and a microsatellite marker were developed that flank the gene at distances of 0.8 and 1.3% recombination, respectively. Received: 19 June 1998 / Accepted: 24 July 1998     

Protein and virus crystal growth on international microgravity laboratory-2.

Two T = 1 and one T = 3 plant viruses, along with a protein, were crystallized in microgravity during the International Microgravity Laboratory-2 (IML-2) mission in July of 1994. The method used was liquid-liquid diffusion in the European Space Agency's Advanced Protein Crystallization Facility (APCF). Distinctive alterations in the habits of Turnip Yellow Mosaic Virus (TYMV) crystals and hexagonal canavalin crystals were observed. Crystals of cubic Satellite Tobacco Mosaic Virus (STMV) more than 30 times the volume of crystals grown in the laboratory were produced in microgravity. X-ray diffraction analysis demonstrated that both crystal forms of canavalin and the cubic STMV crystals diffracted to significantly higher resolution and had superior diffraction properties as judged by relative Wilson plots. It is postulated that the establishment of quasi-stable depletion zones around crystals growing in microgravity are responsible for self-regulated and more ordered growth.     

Tobacco mosaic virus: the first century

Tobacco Mosaic Virus: Pioneering Research for a Century, organized by The Royal Society of Edinburgh, in conjunction with The Royal Society, was held at The Royal Society of Edinburgh, UK, 7–8 August 1998.     

A Survey of German Hops for the Presence of American Hop Latent Virus (AHLV)

The presence of rod-shaped viruses in German hops has been known for a long time and Hop Mosaic Virus (HMV) and Hop Latent Virus (HLV) were proved to be present by serological techniques. When another CARLA-Virus, the American Hop Latent Virus was detected in England an extensive survey was carried out in the German hop growing regions and among wild hops. No natural occurrence of the virus could be detected. Only recently introduced plants of the American clone USDA 21055 in an isolated breeding garden were infected. Spread of the virus to German seedlings could not be detected.     

Observations on a Mixed Soil-Borne Wheat Mosaic Virus and Wheat Spindle Streak Mosaic Virus Infection in Durum Wheat (Triticum durum Desf.)

Both Wheat Spindle Streak Mosaic Virus (WSSMV) and Soil-borne Wheat Mosaic Virus (SBWMV) were found on durum wheat plants (Triticum durum Desf.) grown in a field near Rome (Italy). The simultaneous occurrence of these pathogens was demonstrated by host-symptomatology, pattern of disease occurrence in the field, mechanical transmission tests, as well as by the morphology of viral particles and of ultrastructural modifications. Negatively stained preparates of diseased leaves collected in early, spring showed WSSMV particles and cytoplasmic cylindrical inclusions. SBWMV particles were found only in samples collected later in the season. Ultrathin sections of infected leaves collected in early spring showed characteristic WSSMV modifications such as pinwheels and membranous bodies, whereas samples collected later in the season contained also SBWMV-like crystalline aggregates. WSSMV infection appeared to develop and decline earlier than SBWMV in the leaves of durum wheat plants infected by both, viruses. WSSMV had not been reported in Italy before.     

A DNA polymerase activity is associated with Cauliflower Mosaic Virus

A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa.     

Somaclonal variants resistant to sugarcane mosaic virus and their agronomic characterization

Summary The sugarcane mosaic virus is a pathogen that causes severe disease to sugarcane. New varieties resistant to insects and pathogens have been developed in the last 70 yr through sugarcane breeding programs, but this takes between 10 and 15 yr. Tissue culture techniques are used as an aid for sugarcane improvement to increase desirable agronomic characteristics, such as disease resistance. In the present work, we report the generation of somaclonal variants from sugarcane (Saccharum spp.) cultivar PR62258 susceptible to Sugarcane Mosaic Virus, by somatic embryogenesis. These new variants identified as AT626 and BT627 are resistant to Sugarcanes Mosaic Virus strains A and B, respectively. We established an indirect enzyme linked inmunosorbent assay (ELISA) to test the presence of the viral particles in plants, and its was demonstrated that the leaves of resistant somaclones do not contain viral particles. The field performance of the somaclones AT626 and BT627 was similar to the field performance of the mother plant PR62258.     

Screening promoters for Anthurium transformation using transient expression

Key message

There are multiple publications on Anthurium transformation, yet a commercial product has not been achieved. This may be due to use of non-optimum promoters here we address this problem.

Abstract

Different promoters and tissue types were evaluated for transient β-glucuronidase (GUS) expression in Anthurium andraeanum Hort. ‘Marian Seefurth’ following microprojectile bombardment. Plasmids containing the Ubiquitin 2, Actin 1, Cytochrome C1 from rice, Ubiquitin 1 from maize and 35S promoter from Cauliflower Mosaic Virus fused to a GUS reporter gene were bombarded into in vitro grown anthurium lamina, somatic embryos and roots. The number of GUS foci and the intensity of GUS expression were evaluated for each construct. Ubiquitin promoters from rice and maize resulted in the highest number of expressing cells in all tissues examined. Due to the slow growth of anthurium plants, development of transgenic anthurium plants takes years. This research has rapidly identified multiple promoters that express in various anthurium tissues facilitating the development of transformation vectors for the expression of desirable traits in anthurium plants.