Biochemical and physiological characterization of a new Na+-channel specific peptide from the venom of the Argentinean scorpion Tityus trivittatus

A new peptide with 61 amino acids cross-linked by 4 disulfide bridges, with molecular weight of 6938.12 Da, and an amidated C-terminal amino acid residue was purified and characterized. The primary structure was obtained by direct Edman degradation and sequencing its gene. The peptide is lethal to mammals and was shown to be similar (95% identity) to toxin Ts1 (gamma toxin) from the Brazilian scorpion Tityus serrulatus; it was named Tt1g (from T. trivittatus toxin 1 gamma-like). Tt1g was assayed on several sub-types of Na⁺-channels showing displacement of the currents to more negative voltages, being the hNav1.3 the most affected channel. This toxin displays characteristics typical to the β-type sodium scorpion toxins. Lethality tests and physiological assays indicate that this peptide is probably the most important toxic component of this species of scorpion, known for causing human fatalities in the South American continent.     

Bacterial leaf streak 1 encoding a mitogen-activated protein kinase confers resistance to bacterial leaf streak in rice

Bacterial leaf streak (BLS) is a major bacterial disease of rice. Utilization of host genetic resistance has become one of the most important strategies for controlling BLS. However, only a few resistance genes have been characterized. Previously, a recessive BLS resistance gene bls1 was roughly mapped on chromosome 6. Here, we further delineated bls1 to a 21 kb region spanning four genes. Genetic analysis confirmed that the gene encoding a mitogen-activated protein kinase (OsMAPK6) is the target of the allelic genes BLS1 and bls1. Overexpression of BLS1 weakened resistance to the specific Xanthomonas oryzae pv. oryzicola (Xoc) strain JZ-8, while low expression of bls1 increased resistance. However, both overexpression of BLS1 and low expression of bls1 could increase no-race-specific broad-spectrum resistance. These results indicate that BLS1 and bls1 negatively regulate race-specific resistance to Xoc strain JZ-8 but positively and negatively control broad-spectrum resistance, respectively. Subcellular localization demonstrated that OsMAPK6 was localized in the nucleus. RGA4, which is known to mediate resistance to Xoc, is the potential target of OsMAPK6. Overexpression of BLS1 and low expression of bls1 showed increase in salicylic acid and induced expression of defense-related genes, simultaneously increasing broad-spectrum resistance. Moreover, low expression of bls1 showed increase an in jasmonic acid and abscisic acid, in company with an increase in resistance to Xoc strain JZ-8. Collectively, our study provides new insights into the understanding of BLS resistance and facilitates the development of rice host-resistant cultivars.     

Selection and proliferation of rapid growing cell lines from embryo derived cell cultures of yew tree (Taxus cuspidata Sieb. et Zucc)

Calli were induced from 300,000 embryos isolated from immature to mature stage of seeds collected on late September from 14 elite trees. When the embryos were cultured onto plastic Petri-dish containing 20 mL of modified B5 basal medium supplemented with 3% (w/v) sucrose, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.5% (w/v) polyvinyl polypyrrolidon (PVPP), 2×MS vitamins, 0.5 mg/L gibberellic acid, and 10 mg/L 2,4-D after 2 weeks of culture, yellowish-white calli were immediately formed on the surfaces of embryos, and subcultured for 4 weeks in same culture medium. Because most of calli maintained for more than 3 months were revealed differences in their colors, surface texture, and growth rate, visual selection was made for first round screening. When the size of visually selected calli larger than 19 mm in their diameter were inoculated, persistent proliferation was observed. Among the plating methods tested for the selection of rapid growing cell lines at single cell and/or small cell aggregate level, 2-layer spread plating revealed as the best for single cell cloning. To enhance cell growth and maintain high rate of viability for long-term culture of yew cells in bioreactor, final cell volume less than 50% in SCV seemed to be the best. Time course study revealed that 30% of inoculum density was suitable for fed batch culture. Among the tested conditional media, the rate of 1∶2 (old medium: fresh medium) was recorded at the best for cell growth.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

鸭C4BPα的克隆、表达及其与鸭疫里默氏菌的互作

为研究鸭C4结合蛋白(C4b-binding protein,C4BP)与鸭疫里默氏菌(Riemerella anatipestifer,RA)的相互作用,对鸭C4BPα进行克隆、原核表达,免疫小鼠制备多克隆抗体,并利用间接免疫荧光试验及斑点杂交试验验证C4BP与RA的相互作用。结果显示,鸭C4BPα核苷酸序列全长为1230bp,与鸡C4BPα的相似性最高(82.1%);系统进化树分析发现,鸭C4BPα与鸡C4BPα处于同一系统进化树分支上,两者遗传进化关系最近;C4BPα在大肠杆菌Escherichia coli BL21 (DE3)中能高效表达,重组蛋白以胞内可溶性形式存在;多克隆抗体效价超过1∶10000,并且可以与重组蛋白发生特异性反应;间接免疫荧光试验和斑点杂交试验结果显示RA与鸭C4BP可以发生相互作用。研究结果为进一步揭示RA的致病机制奠定了基础。     

Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from Plasmodium falciparum

The important role of pyruvate kinase during malarial infection has prompted the cloning of a cDNA encoding Plasmodium falciparum pyruvate kinase (pfPyrK), using mRNA from intraerythrocytic-stage malaria parasites. The full-length cDNA encodes a protein with a computed molecular weight of 55.6 kDa and an isoelectric point of 7.5. The purified recombinant pfPyrK is enzymatically active and exists as a homotetramer in its active form. The enzyme exhibits hyperbolic kinetics with respect to phosphoenolpyruvate and ADP, with K_m of 0.19 and 0.12 mM, respectively. pfPyrK is not affected by fructose-1,6-bisphosphate, a general activating factor of pyruvate kinase for most species. Glucose-6-phosphate, an activator of the Toxoplasma gondii enzyme, does not affect pfPyrK activity. Similar to rabbit pyruvate kinase, pfPyrK is susceptible to inactivation by 1 mM pyridoxal-5′-phosphate, but to a lesser extent. A screen for inhibitors to pfPyrK revealed that it is markedly inhibited by ATP and citrate. Detailed kinetic analysis revealed a transition from hyperbolic to sigmoidal kinetics for PEP in the presence of citrate, as well as competitive inhibitory behavior for ATP with respect to PEP. Citrate exhibits non-competitive inhibition with respect to ADP with a K_i of 0.8 mM. In conclusion, P. falciparum expresses an active pyruvate kinase during the intraerythrocytic-stage of its developmental cycle that may play important metabolic roles during infection.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.     

Regulation of the ban gene containing operon of prophage P1

Summary A physical map of the ban gene of P1 and sites relevant to its regulation has been deduced from cloning of the appropriate regions of P1 wild-type and of P1 ban regulatory mutants. The cloning required the presence of P1 repressor in the cell confirming the existence of a repressible ban operon (Austin et al. 1978). Evidence for additional member(s) of that operon is presented. Of particular interest for understanding the regulation of ban are the relative positions of a binding site for the P1 repressor and of the regulatory mutations bac and crr that render ban expression constitutive. The results reveal a repressible operon-like structure of about 4 kb within the P1 EcoRI-3 fragment that comprises a c1 repressor binding site/bac additional gene(s) — crr/ban in the clockwise direction of the circular map of P1.