The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

Secretion, purification, and characterization of a recombinant Aspergillus oryzae tannase in Pichia pastoris

Tannase (tannin acyl hydrolase) is an industrially important enzyme produced by a large number of fungi, which hydrolyzes the ester and depside bonds of gallotannins and gallic acid esters. In the present work, a tannase from Aspergillus oryzae has been cloned and expressed in Pichia pastoris. The catalytic activity of the recombinant enzyme was assayed. A secretory form of enzyme was made with the aid of Saccharomyces cerevisiae alpha-factor, and a simple procedure purification protocol yielded tannase in pure form. The productivity of secreted tannase achieved 7000 IU/L by fed-batch culture. Recombinant tannase had a molecular mass of 90 kDa, which consisted of two kinds of subunits linked by a disulfide bond(s). Our study is the first report on the heterologous expression of tannase suggesting that the P. pastoris system represents an attractive means of generating large quantities of tannase for both research and industrial purpose.     

Megator, an essential coiled-coil protein that localizes to the putative spindle matrix during mitosis in Drosophila

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.     

Improving the performance of DomainParser for structural domain partition using neural network

Structural domains are considered as the basic units of protein folding, evolution, function and design. Automatic decomposition of protein structures into structural domains, though after many years of investigation, remains a challenging and unsolved problem. Manual inspection still plays a key role in domain decomposition of a protein structure. We have previously developed a computer program, DomainParser, using network flow algorithms. The algorithm partitions a protein structure into domains accurately when the number of domains to be partitioned is known. However the performance drops when this number is unclear (the overall performance is 74.5% over a set of 1317 protein chains). Through utilization of various types of structural information including hydrophobic moment profile, we have developed an effective method for assessing the most probable number of domains a structure may have. The core of this method is a neural network, which is trained to discriminate correctly partitioned domains from incorrectly partitioned domains. When compared with the manual decomposition results given in the SCOP database, our new algorithm achieves higher decomposition accuracy (81.9%) on the same data set.     

TAMS technology for simple and efficient in vitro site-directed mutagenesis and mutant screening

Site-directed mutagenesis is an invaluable tool for functional studies and genetic engineering. However, most current protocols require the target DNA to be cloned into a plasmid vector before mutagenesis can be performed, and none of them are effective for multiple-site mutagenesis. We now describe a method that allows mutagenesis on any DNA template (eg. cDNA, genomic DNA and plasmid DNA), and is highly efficient for multiple-site mutagenesis (up to 100%). The technology takes advantage of the requirement that, in order for DNA polymerases to elongate, it is crucial that the 3′ sequences of the primers match the template perfectly. When two outer mutagenic oligos are incorporated together with the desired mutagenic oligos into the newly synthesised mutant strand, they serve as anchors for PCR primers which have 3′ sequences matching the mutated nucleotides, thus amplifying the mutant strand only. The same principle can also be used for mutant screening.     

A new mode of Ca2+ signaling by G protein-coupled receptors: gating of IP3 receptor Ca2+ release channels by Gbetagamma

The most common form of Ca(2+) signaling by Gq-coupled receptors entails activation of PLCbeta2 by Galphaq to generate IP(3) and evoke Ca(2+) release from the ER. Another form of Ca(2+) signaling by G protein-coupled receptors involves activation of Gi to release Gbetagamma, which activates PLCbeta1. Whether Gbetagamma has additional roles in Ca(2+) signaling is unknown. Introduction of Gbetagamma into cells activated Ca(2+) release from the IP(3) Ca(2+) pool and Ca(2) oscillations. This can be due to activation of PLCbeta1 or direct activation of the IP(3)R by Gbetagamma. We report here that Gbetagamma potently activates the IP(3) receptor. Thus, Gbetagamma-triggered [Ca(2+)](i) oscillations are not affected by inhibition of PLCbeta. Coimmunoprecipitation and competition experiments with Gbetagamma scavengers suggest binding of Gbetagamma to IP(3) receptors. Furthermore, Gbetagamma inhibited IP(3) binding to IP(3) receptors. Notably, Gbetagamma activated single IP(3)R channels in native ER as effectively as IP(3). The physiological significance of this form of signaling is demonstrated by the reciprocal sensitivity of Ca(2+) signals evoked by Gi- and Gq-coupled receptors to Gbetagamma scavenging and PLCbeta inhibition. We propose that gating of IP(3)R by Gbetagamma is a new mode of Ca(2+) signaling with particular significance for Gi-coupled receptors.     

A Rapid and Simple PCR-based Method for Analysis of Transgenic Fish using a Restricted Amount of Fin Tissue

The protocol described in this paper offers a simple and rapid method for PCR analysis of transgenes using a restricted amount of fin tissue from small-sized transgenic fish. A simple preparation of fin lysate using a buffer containing a low concentration of an ionic detergent, SDS (0.01%), followed by neutralization with a second buffer containing higher concentrations of non-ionic detergents NP40 (2%) and Tween 20 (2%) consistently provides a reliable quantity of high-quality DNA template for PCR amplification of transgenes. Based on this protocol, transgenic fish can be clearly distinguished from non-transgenic fish using PCR in a rapid and reproducible manner. Tedious DNA purifications are avoided while fidelity of amplification and efficient identification of transgenic fish are maintained.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Anti-tumor activity of the farnesyl-protein transferase inhibitors arteminolides, isolated from Artemisa

Members of the Artemisia genus are important medicinal plants found throughout the world. Arteminolides A-D (1-4), isolated from the aerial parts of Artemisia, have an inhibitory activity on farnesyl-protein transferase (FPTase; EC 2.5.1.29) in in vitro assay. This study was carried out with the purpose of validating anti-tumor effects of the compounds in human tumor cells and mouse xenograft model. The arteminolides inhibited tumor cell growth in a dose-dependent manner. Furthermore, arteminolide C (3) blocked in vivo growth of human colon and lung tumor xenograft without the loss of body weight in nude mice.