181 research outputs found
Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm
A new glutelin gene, designated GluD-1, has been discovered by comparing the seed storage proteins from 48 japonica and indica rice cultivars on SDS-PAGE gels. Evidence that GluD-1 is a member of the glutelin family was provided by Western blots using anti-glutelin antiserum and by mapping the gene to the chromosomal glutelin gene cluster. The limited GluD-1 size polymorphism among the rice varieties is due to amino acid substitutions rather than to post-transcriptional modification. GluD-1 is maximally expressed in the starchy endosperm starting at 5 d after flowering (DAF) and increasing through 30 DAF, a major difference from the other glutelins which are primarily expressed in the subaleurone from 10–16 DAF. Only about 0.2 kb of the GluD-1 promoter was sufficient to confer inner starchy endosperm-specific expression. The 0.2 kb truncated GluD-1 promoter contains a bifactorial endosperm box consisting of a truncated GCN4 motif (TGA(G/C)TCA) and AAAG Prolamin box (P box), and ACGT and AACA motifs as cis-regulatory elements. Gel retardation assays and trans-activation experiments indicated that the truncated GCN4 and P box are specifically recognized by RISBZ1 b-ZIP and RPBF Dof activators in vitro, respectively, and are synergistically transactivated, indicating that combinatorial interactions of these motifs are involved in essential endosperm-specific regulation. Furthermore, deviation from the cognate GCN4 motif alters tissue-specific expression in the inner starchy endosperm to include other endosperm tissues
Seed-specific expression of truncated OsGAD2 produces GABA-enriched rice grains that influence a decrease in blood pressure in spontaneously hypertensive rats
Gamma-aminobutyric acid (GABA) is a four-carbon amino acid that is commonly present in living organisms and functions as a major inhibitory neurotransmitter in mammals. It is understood to have a potentially anti-hypertensive effect in mammals. GABA is synthesized from glutamate by glutamate decarboxylase (GAD). In plants, GAD is regulated via its calmodulin-binding domain (CaMBD) by Ca2+/CaM. We have previously reported that a C-terminal truncated version of one of the five rice GAD isoforms, GAD2ΔC, revealed higher enzymatic activity in vitro and that its over-expression resulted in exceptionally high GABA accumulation (Akama and Takaiwa, J Exp Bot 58:2699–2607, 2007). In this study, GAD2ΔC, under the control of the rice glutelin promoter (GluB-1), was introduced into rice cells via Agrobacterium-mediated transformation to produce transgenic rice lines. Analysis of the free amino acid content of rice grains revealed up to about a 30-fold higher level of GABA than in non-transformed rice grains. There were also very high levels of various free protein amino acids in the seeds. GABA-enriched rice grains were milled to a fine powder for oral administration to spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs). Six weeks of administration showed that transgenic rice brings about a 20 mmHg decrease in blood pressure in two different kinds of SHRs, while there was no significant hypotensive effect in WKYs. These results suggest an alternative way to control and/or cure hypertension in humans with GABA-enriched rice as part of a common daily diet
Analysis of the modes of energy consumption of the complex of an incoherent scattering of the institute of ionosphere of national academy of sciences and the ministry of education and science of Ukraine
У даній статті представлені результати аналізу режимів енергоспоживання комплексу некогерентного розсіяння Інституту іоносфери НАН і МОН України з метою вирішення проблеми підвищення енергоефективності науково-дослідного комплексу та створення енергоефективної системи електропостачання, яка забезпечить стійку роботу наукового обладнання для виконання дослідницьких програм НАН України. Описана система електроживлення комплексу та режими енергоспоживання комплексу. Описано пристрої радарної системи, а також найбільш потужні споживачі електроенергії, які споживають електроенергію на експериментальні і господарські потреби. Проаналізовано енергоспоживання комплексу некогерентного розсіяння за 2013 р. Отримано і представлено графіки середньої споживаної потужності (середньодобовий показник) і середньої споживаної потужності в режимі вимірювань. Описана доцільність проведення робіт з оптимізації енергопостачання науково-дослідного комплексу Інституту іоносфери. Запропоновано можливі заходи для зниження економічної вартості проведення експериментів з дослідження іоносфери науково-дослідного комплексу некогерентного розсіяння. Проведено аналіз робіт сучасних авторів з метою показати, що підвищення ефективності функціонування систем електропостачання є актуальною проблемою сучасних досліджень.This article presents the results of the analysis of the energy consumption modes of the incoherent scattering complex of the Institute of Ionosphere of the National Academy of Sciences and the Ministry of Education and Science of Ukraine to solve the problem of increasing the energy efficiency of a research complex and creating an energy efficient power supply system that will ensure the sustainability of scientific equipment for research programs
of the National Academy of Sciences of Ukraine. The system of power supply of the complex and modes of power consumption of the complex are described. The devices of the radar system are described, as well as the most powerful consumers of electricity, which consume electricity for experimental and economic needs. The energy consumption of the incoherent scattering complex in 2013 is analyzed. Graphs of the average power consumption (daily average) and average power consumption in measurement modes were obtained and presented. The feasibility of work to optimize the energy supply of the research complex of the institute of the ionosphere is described. Possible measures are proposed to reduce the economic cost of conducting experiments on the study of the ionosphere of an incoherent scattering research complex. The analysis of the works of modern authors i s
carried out in order to show that increasing the efficiency of the power supply systems is an actual problem of modern research
A Cost-Effective ELP-Intein Coupling System for Recombinant Protein Purification from Plant Production Platform
BACKGROUND: Plant bioreactor offers an efficient and economical system for large-scale production of recombinant proteins. However, high cost and difficulty in scaling-up of downstream purification of the target protein, particularly the common involvement of affinity chromatography and protease in the purification process, has hampered its industrial scale application, therefore a cost-effective and easily scale-up purification method is highly desirable for further development of plant bioreactor. METHODOLOGY/PRINCIPAL FINDINGS: To tackle this problem, we investigated the ELP-intein coupling system for purification of recombinant proteins expressed in transgenic plants using a plant lectin (PAL) with anti-tumor bioactivity as example target protein and rice seeds as production platform. Results showed that ELP-intein-PAL (EiP) fusion protein formed novel irregular ER-derived protein bodies in endosperm cells by retention of endogenous prolamins. The fusion protein was partially self-cleaved in vivo, but only self-cleaved PAL protein was detected in total seed protein sample and deposited in protein storage vacuoles (PSV). The in vivo uncleaved EiP protein was accumulated up to 2-4.2% of the total seed protein. The target PAL protein could be purified by the ELP-intein system efficiently without using complicated instruments and expensive chemicals, and the yield of pure PAL protein by the current method was up to 1.1 mg/g total seed protein. CONCLUSION/SIGNIFICANCE: This study successfully demonstrated the purification of an example recombinant protein from rice seeds by the ELP-intein system. The whole purification procedure can be easily scaled up for industrial production, providing the first evidence on applying the ELP-intein coupling system to achieve cost-effective purification of recombinant proteins expressed in plant bioreactors and its possible application in industry
Role of Conserved Non-Coding Regulatory Elements in LMW Glutenin Gene Expression
Transcriptional regulation of LMW glutenin genes were investigated in-silico, using publicly available gene sequences and expression data. Genes were grouped into different LMW glutenin types and their promoter profiles were determined using cis-acting regulatory elements databases and published results. The various cis-acting elements belong to some conserved non-coding regulatory regions (CREs) and might act in two different ways. There are elements, such as GCN4 motifs found in the long endosperm box that could serve as key factors in tissue-specific expression. Some other elements, such as the AACA/TA motifs or the individual prolamin box variants, might modulate the level of expression. Based on the promoter sequences and expression characteristic LMW glutenin genes might be transcribed following two different mechanisms. Most of the s- and i-type genes show a continuously increasing expression pattern. The m-type genes, however, demonstrate normal distribution in their expression profiles. Differences observed in their expression could be related to the differences found in their promoter sequences. Polymorphisms in the number and combination of cis-acting elements in their promoter regions can be of crucial importance in the diverse levels of production of single LMW glutenin gene types
Novel Vaccines to Human Rabies
Rabies, the most fatal of all infectious diseases, remains a major public health problem in developing countries, claiming the lives of an estimated 55,000 people each year. Most fatal rabies cases, with more than half of them in children, result from dog bites and occur among low-income families in Southeast Asia and Africa. Safe and efficacious vaccines are available to prevent rabies. However, they have to be given repeatedly, three times for pre-exposure vaccination and four to five times for post-exposure prophylaxis (PEP). In cases of severe exposure, a regimen of vaccine combined with a rabies immunoglobulin (RIG) preparation is required. The high incidence of fatal rabies is linked to a lack of knowledge on the appropriate treatment of bite wounds, lack of access to costly PEP, and failure to follow up with repeat immunizations. New, more immunogenic but less costly rabies virus vaccines are needed to reduce the toll of rabies on human lives. A preventative vaccine used for the immunization of children, especially those in high incidence countries, would be expected to lower fatality rates. Such a vaccine would have to be inexpensive, safe, and provide sustained protection, preferably after a single dose. Novel regimens are also needed for PEP to reduce the need for the already scarce and costly RIG and to reduce the number of vaccine doses to one or two. In this review, the pipeline of new rabies vaccines that are in pre-clinical testing is provided and an opinion on those that might be best suited as potential replacements for the currently used vaccines is offered
Processing of chloroplast ribosomal RNA transcripts in Euglena gracilis bacillaris
The ribosomal RNA operons ( rrn operons) of Euglena gracilis chloroplasts contain genes for (in order) 16S rRNA, tRNA Ile , tRNA Ala , 23S rRNA and 5S rRNA. Major sites of cleavage of the primary rrn transcript were identified by Northern blot hybridization and S1-mapping. The presumptive termini of all of the mature products have now been identified. During initial processing in the chloroplast, the primary transcript is cleaved between the two tRNAs and between the 23S and 5S rRNAs so as to separate the sequences found in the different mature rRNAs. Subsequently the tRNAs are separated from the rRNAs, further trimming provides the remaining proper ends, and the 3′-ends of the tRNAs are added.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46969/1/294_2004_Article_BF00419917.pd
Upstream regulatory architecture of rice genes: summarizing the baseline towards genus-wide comparative analysis of regulatory networks and allele mining
Synergism between RPBF Dof and RISBZ1 bZIP Activators in the Regulation of Rice Seed Expression Genes
The Dof (DNA binding with one finger) transcriptional activator rice (Oryza sativa) prolamin box binding factor (RPBF), which is involved in gene regulation of rice seed storage proteins, has been isolated from rice cDNA expressed sequence tag clones containing the conserved Dof. RPBF is found as a single gene per haploid genome. Comparison of RPBF genomic and cDNA sequences revealed that the genomic copy is interrupted by one long intron of 1,892 bp in the 5′ noncoding region. We demonstrated by transient expression in rice callus protoplasts that the isolated RPBF trans-activated several storage protein genes via an AAAG target sequence located within their promoters, and with methylation interference experiments the additional AAAG-like sequences in promoters of genes expressed in maturing seeds were recognized by the RPBF protein. Binding was sequence specific, since mutation of the AAAG motif or its derivatives decreased both binding and trans-activation by RPBF. Synergism between RPBF and RISBZ1 recognizing the GCN4 motif [TGA(G/C)TCA] was observed in the expression of many storage protein genes. Overexpression of both transcription factors gave rise to much higher levels of expression than the sum of individual activities elicited by either RPBF or RISBZ1 alone. Furthermore, mutation of recognition sites suppressed reciprocal trans-activation ability, indicating that there are mutual interactions between RISBZ1 and RPBF. The RPBF gene is predominantly expressed in maturing endosperm and coordinately expressed with seed storage protein genes, and is involved in the quantitative regulation of genes expressed in the endosperm in cooperation with RISBZ1
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