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Review: Plant Binary Vectors of Ti Plasmid in Agrobacterium tumefaciens with a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1  [PDF]
Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2013, DOI: 10.4236/ajps.2013.44115

This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli. A general trend in the binary vector development has been to increase the plasmid stability during a long co-cultivation period of A. tumefaciens with the target host plant tissues. A second trend is to understand the molecular mechanism of broad host-range replication, and to use it to reduce the size of plasmid for ease in cloning and for higher plasmid yield in E. coli. The broad host-range replicon of VS1 was shown to be a choice of replicon over those of pRK2, pRi and pSA because of the superior stability and of small well-defined replicon. Newly developed plant binary vectors pLSU has the small size of plasmid backbone (4566 bp) consisting of VS1 replicon (2654 bp), ColE1 replicon (715 bp), a bacterial kanamycin (999 bp) or tetracycline resistance gene,

Review: Plant Growth Hormone Cytokinins Control the Crop Seed Yield  [PDF]
Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2014, DOI: 10.4236/ajps.2014.514231

This review chronicles the development of the cytokinin research during the last 30 years. Cytokinin and auxin are the two major plant growth hormones that control virtually all aspects of growth and development in higher plants. The pathways for cytokinin biosynthesis and metabolism have been characterized by the identification of isopentenyl pyrophosphate transferase, cytokinin oxidases, cytokinin hydroxylase, zeatin cis-/trans-isomerase, cytokinin phosphoribosyl hydrolases, cytokinin-specific riboside phosphorylase, and others enzymes. Loss-of function mutant phenotypes of cytokinin degradation/activating enzymes indicate the regulation of concentration and spatial distribution of bio-active cytokinin plays a pivotal role in the increase in panicle size, in the numbers of floral organs, and eventually in seed yield. One of the most fundamental questions in the cytokinin field is one concerning the prevalence of cis-zeatin in monocotyledonous crops (rice and maize) and in dicotyledonous legumes (pea, chickpea) and potato/sweet potato. A hypothesis is that cis-zeatin is synthesized by the cis-specific hydroxylation of the terminal methyl group of N6-isopentenyl side
chain of

Review: Plant Genome Editing Targeted by RNA-Guided DNA Endonuclease CRISPR/Cas9  [PDF]
Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2017, DOI: 10.4236/ajps.2017.813233
Abstract: This review chronicles the development of the research on CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR associated protein 9) during the last 30 years from the discovery of CRISPR sequence, of biological significance and of the molecular mechanism for adaptive bacterial immunity. It describes recent works on structural and functional diversity of CRISPR/Cas systems, and on three-dimensional structure-based improvements of on-target specificity of CRISPR/Cas9 and Cpf1 endonucleases. The review ends with the application of CRISPR/Cas9 to targeted editing of plant genomes. Importantly, plant commodities modified by CRISPR-Cas9 have not been considered as genetically modified organisms (GMO) as long as foreign DNAs from plant pests were not introduced, according to the recent determination by the USDA.
Low Co-Cultivation Temperature at 20°C Resulted in the Reproducible Maximum Increase in Both the Fresh Weight Yield and Stable Expression of GUS Activity after Agrobacterium tumefaciens-Mediated Transformation of Tobacco Leaf Disks  [PDF]
Guiying Su, Sunjung Park, Seokhyun Lee, Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2012, DOI: 10.4236/ajps.2012.34064
Abstract: The importance of controlled temperature during the four-days co-cultivation period was evaluated under the most physiologically relevant conditions for Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cv. Xanthi (nn, Smith)) leaf disks. We compared the effect of temperatures ranging from 15°C, 18°C, 20°C, 22°C to 25°C on the stable expression of β-glucuronidase (GUS) activity of 14 days old hygromycin-selected leaf disks, and on the increase in the fresh weight yield of 28 days old kanamycin-selected calli. The highest average of GUS activity was obtained at 20°C among the five temperatures tested although the difference between the 18°C and 20°C treatment was not statistically significant. The GUS activity at 15°C was statistically lower than those at 18°C and 20°C. The GUS activity in 22°C treatment was an intermediate between the highest (18/20°C) and second highest averages (15°C), and was not statistically significantly different. The lowest average of GUS activity was observed at 25°C. The highest increase in the plate average of fresh weight yield was obtained at 20°C among the five temperature tested. The 20°C treatment was statistically significantly better than the 15°C and 18°C treatments. The 20°C co-cultivation treatment resulted in the higher FW yield than 22°C and 25°C even though the differences were not statistically significant. In conclusion, low co-cultivation temperature at 20°C resulted in the reproducible maximum increase in both the fresh weight yield and stable expression of GUS activity after transformation of tobacco leaf disks.
Tetracycline-Based Binary Ti Vectors pLSU with Efficient Cloning by the Gateway Technology for Agrobacterium tumefaciens-Mediated Transformation of Higher Plants  [PDF]
Seokhyun Lee, Guiying Su, Eric Lasserre, Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2013, DOI: 10.4236/ajps.2013.47173
Abstract: We constructed small high-yielding binary Ti vectors with a bacterial tetracycline resistance gene to facilitate efficient cloning afforded by the Gateway Technology (Invitrogen) for Agrobacterium tumefaciens-mediated transformation of higher plants. The Gateway Technology vectors are kanamycin-based, thus tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the Escherichia coli media. We reduced the size of the tetracycline resistance gene TetC from pBR322 to 1468 bp containing 1191 bp of the coding region, 93 bp of 5’-upstream, and 184 bp 3’-downstream region. The final size of binary Ti vector skeleton pLSU11 is 5034 bp. pLSU12 and 13 have the kanamycin resistance NPTII gene as a plant-selectable marker. pLSU13 and 15 contain the hygromycin resistance HPH gene as a selection marker. pLSU13 and 15 also have the β-glucuronidase (GUS) reporter gene in addition to the plant selection marker. We also constructed a mobilizable version of tetracycline-based binary Ti vector pLSU16 in which the mob function of ColE1 replicon was maintained for mobilization of the binary vector from E. coli to A. tumefaciens by tri-parental mating. The final size of binary Ti vector skeleton pLSU16 is 5580 bp. New tetracycline- based binary Ti vectors pLSU12 were found as effective as kanamycin-based vector pLSU2 in promoting a 10-fold increase in fresh weight yield of kanamycin-resistant calli after A. tumefaciens-mediated transformation of tobacco leaf discs. Using the Gateway Technology we introduced the plant-expressible GUSgene to the T-DNA of binary Ti vector pLSU12. Expression of the β-glucuronidase enzyme activity was demonstrated by histochemical staining of the GUS activity in transformed tobacco leaf discs.
Modified Bean Seed Protein Phaseolin Did Not Accumulate Stably in Transgenic Tobacco Seeds after Methionine Enhancement Mutations  [PDF]
Eric Lasserre, T. S. Ko, John M. Dyer, Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2015, DOI: 10.4236/ajps.2015.65069
Abstract: The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in methionine, an essential amino acid for human and animal health. To improve the nutritional quality of common bean, we designed methionine enhancement of phaseolin based on the three-dimensional structure of protein, de novo design principles and genetic information. Amino acid substitution and loop insertion were targeted to the interior and exterior, respectively, of the protein’s β-barrels. First, we introduced the methionine enhancement mutations into phaseolin cDNA, expressed cDNA in Escherichia coli and purified monomeric non-glycosylated proteins. Biophysical analysis of E. coli-expressed proteins demonstrated a similar structural stability of wild-type and mutant phaseolin monomers. Here, we attempted to test the structural stability of the methionine-enhanced phaseolin by introducing phaseolin cDNA to tobacco via Agrobacterium tumefaciens-mediated transformation of leaf disks, regenerating transgenic tobacco plants, and examining the accumulation of phaseolin protein in mature transgenic tobacco seeds. We used seven constructs containing different extents of methionine enhancement, ranging from the original 3 to maximum 33 methionines per 397 amino acid residues. ELISA analyses indicated that the methionine-enhanced phaseolins did not accumulate as stably in mature transgenic tobacco seeds as the wild-type phaseolin. It seems likely that the methionine-enhanced phaseolin proteins were under the stringent scrutiny of the protein quality control mechanism in the endoplasmic reticulum (ER), Golgi complex and/or vacuolar protein bodies. The protein degradation is probably to occur in the vacuolar protein bodies due to the instability of the trimer assembly caused by the methionine enhancement mutations targeting either amino-acids substitutions or/and loop insertions to the interior β-sheets and tum/loop regions, respectively, of N- and C-barrel structures.
Sweet Potato Leaf Curl Virus: Coat Protein Gene Expression in Escherichia coli and Product Identification by Mass Spectrometry  [PDF]
Dina Lida Gutierrez Reynoso, Rodrigo A. Valverde, Norimoto Murai
American Journal of Plant Sciences (AJPS) , 2015, DOI: 10.4236/ajps.2015.619296
Abstract: Sweet potato is one of the first natural GMOs, genetically modified 8000 years ago by Agrobacterium rhizogenes as reported recently by Kyndt et al. A section of 10 kbp long DNA (Transferred- DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizo-genes and has been maintained in all 291 hexaploid sweet potato cultivars of the world. The maintenance in the sweet potato genome and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3-acetamide hydrolase (iaaH) are likely to be physiologically significant since these enzymes convert tryptophan to indole-3-acetic acid, a major plant growth hormone auxin. Sweet potato (Ipomoea batatas (L.) Lam) is ranked the third most important root crop after potato and cassava, and the seventh in global food crop production with more than 126 million metric tons. Although sweet potato originated in Central or South America, China currently produces over 86% of world production with 109 million metric tons. In the United States, North Carolina is the leading producer with 38.5% of the 2007 sweet potato production, followed by California, Mississippi, and Louisiana with 23%, 19%, and 15.9%, respectively. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) belonging to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, one of the most predominant sweet potato cultivars in the US, it can reduce the yield up to 26%. Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strain XL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 μg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltosebinding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of SPLCV based on the fact that the mass spectrometry elucidated the sequences corresponding to 37% of amino acid positions of the
The Role of Lipid Rafts in Cancer Cell Adhesion and Migration
Toshiyuki Murai
International Journal of Cell Biology , 2012, DOI: 10.1155/2012/763283
Abstract: Lipid rafts are cholesterol-enriched microdomains of the cell membrane and possess a highly dynamic nature. They have been involved in various cellular functions including the regulation of cell adhesion and membrane signaling through proteins within lipid rafts. The dynamic features of the cancer cell surface may modulate the malignant phenotype of cancer, including adhesion disorders and aggressive phenotypes of migration and invasion. Recently, it was demonstrated that lipid rafts play critical roles in cancer cell adhesion and migration. This article summarizes the important roles of lipid rafts in cancer cell adhesion and migration, with a focus on the current state of knowledge. This article will improve the understanding of cancer progression and lead to the development of novel targets for cancer therapy. 1. Introduction The alternation of cell adhesion and highly migratory behavior are the most prominent features of cancer cells, and play critical roles in their aggressive invasion and metastatic spread [1]. These processes appear to be facilitated by remodeling of the extracellular matrix (ECM) of the tumor microenvironment and adhesion molecules at the cancer cell surface and affected by both the interaction between ECM and adhesion molecules and by growth factor signaling [2, 3]. The proteolytic ectodomain cleavage and release (shedding) of adhesion molecules are also critical regulatory steps in cancer cell adhesion and migration [4, 5]. To date, cholesterol-enriched membrane microdomains called “lipid rafts” have been implicated in a variety of pathogeneses [6]; neurological diseases including Alzheimer’s [7], Parkinson’s [8], and prion diseases [9]; cardiovascular diseases; immune disorders such as systemic lupus erythematosus [10] and HIV infection [11]. Lipid rafts have been also implicated in signaling pathways in cancer progression [12], but how these microdomains affect the adhesion and migration of invasive cancer cells remains obscure. In this paper, recent findings on the roles of lipid rafts in cancer cell adhesion and migration will be reviewed. 2. Lipid Raft Structure The prevailing model of cellular membrane structure was proposed by Singer and Nicolson, and this model is known as the fluid mosaic model, where globular proteins float in a lipid bilayer with a basic structure [13]. Later, the model was improved by Simons and van Meer, who suggested the existence of microdomains or “rafts” in the plasma membrane of epithelial cells [14]. In the current understanding of the lipid raft model, cholesterol- and sphingolipid-enriched
Lezioni dal grande terremoto e dallo tsunami del marzo 2011 in Giappone orientale
Shunji Murai
GEOmedia , 2012,
Abstract: Questo rapporto riassume ciò che abbiamo imparato dal disastro del Gran Terremoto e Maremoto nell’Est del Giappone che avvenne l’11 marzo 2011 su una vasta area. Include le descrizioni delle perdite dovute al disastro, le lezioni dai disastri passati e presenti, un focus su ciò che è stato fatto correttamente e cosa è andato storto, l'incidente nucleare della Centrale Nucleare di Fukushima (NPS, Nuclear Power Station) e le opinioni dell’autore. Lessons from the Disaster of East Japan Great Earthquake and Tsunami 311 The report summarizes what we have learnt from the disaster of the East Japan Great Earthquake and Tsunami which oc-curred on March 11, 2011 over a wide area of East Japan. It includes descriptions of the losses due to the disaster, lessons from the past and present disasters with focus on what was done correctly and what went wrong, the accident at Fuku-shima Nuclear Power Station (NPS) and my views.
Gaku, Jutsu and Gei in Music Therapy
Yasuji Murai
Voices: A World Forum for Music Therapy , 2001,
Abstract: Needless to say, every therapist is trying hard to create his/her own professional personality in order to become a good therapist as a life long task. And the only premise for this task is to be a specialist of music therapy. However, aiming to be a good therapist varies depending on where each person puts his/her basis. For example, since my major work has been around schizophrenia, I have been trying to watch schizophrenic people closely and constantly. It might seem to be a natural task for a music therapist who deals with schizophrenia. But every time I have tried to summarize this disease, I have felt keenly how challenging this task is, even after a 30- year career. What on the earth is the cause of schizophrenia? It is said that the person's own make-up or hereditary predisposition might come into control, or the environmental factors might operate. But how do these things build up an individual to schizophrenia? How and when are the so-called "pre-personality tendencies" made, such as withdrawing or lack of self-confidence? This pattern of thoughts/behaviors that entice the person to the world of morbid experiences, has a close connection with the build-up of the disease. I personally believe that in this secret connection, there are some clues to the treatment. The work of discovering these clues is achieved by reviewing my own past clinical cases and other researchers' papers.
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