A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes

The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.     

Clinicopathological and immunological studies on toxoids vaccine as a successful alternative in controlling clostridial infection in broilers

The present study was conducted to evaluate the efficacy and safety of three vaccination regimes of Clostridium perfringens (C. perfringens) type A, C and combined A&C toxoids based on their clinical signs and immunological effects. The vaccines were administered two times at two weeks interval (7 & 21 days old), then the birds were challenged (35 days old) with virulent strains of C. perfringens type A, C and combined A&C. Blood samples were taken one week after the first and second vaccination as well as after challenge. The evaluated parameters in this study included: clinical signs, gross intestinal lesions, complete blood count (CBC), serum protein, liver profiles, and enzyme-linked immunosorbent assay (ELISA) test for detecting serum antibody titers. The results revealed that immunization of broilers with C. perfringens type A, C and combined A&C toxoids resulted in a significant decrease in numbers of chickens with intestinal lesions particularly with the A&C toxoids vaccine. Results of the CBC values were significantly increased in all treated groups and challenged groups. Total leukocytic count decreased in challenged non vaccinated group while increased in challenged vaccinated birds. Results of biochemical assays implicated that there were a significant increase in serum protein and liver profiles. ELISA results explored a significant increase in antibody titers after immunization of broilers with C. perfringens type A, C and combined A&C toxoids particularly after the second dose of vaccination. We concluded that immunization of broilers with toxoid vaccines particularly the combined type A & C is safe, well-tolerated and can protect broiler chickens against necrotic enteritis particularly after the second booster dose of the vaccine.     

Sterile Inflammation in Acetaminophen-induced Liver Injury Is Mediated by Cot/tpl2

Cot/tpl2 (MAP3K8) activates MKK1/2-Erk1/2 following stimulation of the Toll-like/IL-1 receptor superfamily. Here, we investigated the role of Cot/tpl2 in sterile inflammation and drug-induced liver toxicity. Cot/tpl2 KO mice exhibited reduced hepatic injury after acetaminophen challenge, as evidenced by decreased serum levels of both alanine and aspartate aminotransferases, decreased hepatic necrosis, and increased survival relative to Wt mice. Serum levels of both alanine and aspartate aminotransferases were also lower after intraperitoneal injection of acetaminophen in mice expressing an inactive form of Cot/tpl2 compared with Wt mice, suggesting that Cot/tpl2 activity contributes to acetaminophen-induced liver injury. Furthermore, Cot/tpl2 deficiency reduced neutrophil and macrophage infiltration in the liver of mice treated with acetaminophen, as well as their hepatic and systemic levels of IL-1α. Intraperitoneal injection of damage-associated molecular patterns from necrotic hepatocytes also impaired the recruitment of leukocytes and decreased the levels of several cytokines in the peritoneal cavity in Cot/tpl2 KO mice compared with Wt counterparts. Moreover, similar activation profiles of intracellular pathways were observed in Wt macrophages stimulated with Wt or Cot/tpl2 KO damage-associated molecular patterns. However, upon stimulation with damage-associated molecular patterns, the activation of Erk1/2 and JNK was deficient in Cot/tpl2 KO macrophages compared with their Wt counterparts; an effect accompanied by weaker release of several cytokines, including IL-1α, an important component in the development of sterile inflammation. Taken together, these findings indicate that Cot/tpl2 contributes to acetaminophen-induced liver injury, providing some insight into the underlying molecular mechanisms.     

一种构建马链球菌兽疫亚种血红素受体基因缺失突变株的方法

【目的】探讨一种构建马链球菌兽疫亚种基因缺失突变株的方法。【方法】PCR扩增目的基因,用pJR700温度敏感载体系统,构建目的基因载体;反向PCR扩增目的基因缺失载体片段,连接,产生目的基因缺失载体;电转化缺失重组质粒导入感受态细胞,先在37℃卡拉霉素(kan)培养基中连续培养,然后在30℃不含kan液体培养基中传代,挑取抗生素敏感菌落,PCR扩增检测抗生素敏感菌染色体上目的基因片段和链黑霉素抗性实验确认血红素受体基因缺失。【结果】获得不含抗生素基因的马链球菌兽疫亚种血红素受体基因缺失突变株。【结论】用pJR700温度敏感载体系统,构建马链球菌兽疫亚种基因缺失突变株是可行的。     

芽变毛白杨形态解剖和同功酶初步分析

目的:为了解芽变毛白杨(以下称芽变杨)新品种的特点及融合亲本。方法:试验以芽变杨、毛白杨和大官杨为研究对象,进行形态学、同功酶等分析。结果:在外部形态上芽变杨既有毛白杨和大官杨的特点,具有两亲本折中的形态。显微观察,导管壁较厚,管腔占木质部的比例小。同功酶谱带具有两亲本特有的条带,这为芽变杨是大官杨和毛白杨两者的融合突变提供了直接证据。     

Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.     

Membrane structure of the human immunodeficiency virus gp41 fusion peptide by molecular dynamics simulation: II. The glycine mutants

In this work, molecular dynamics (MD) simulation of the interaction of three mutants, G3V, G5V and G10V, of the human immunodeficiency virus (HIV) gp41 16-residue fusion peptide (FP) with an explicit palmitoyloleoylphosphatidyl-ethanolamine (POPE) lipid bilayer was performed. The goals of this work are to study the correlation of the fusogenic activity of the FPs with the mode of their interaction with the bilayer and to examine the roles of the many glycine residues in the FP in the fusion process. The results of this work corroborate the main conclusion of our earlier MD work of the WT FP and several mutants with polar substitution. These two studies provide correlation between the mode of insertion and the fusogenic activity of these peptides and support the hypothesis that an oblique insertion of the fusion domain of the viral protein is required for fusogenic activity. Inactive mutants interact with the bilayer by a surface-binding mode. The results of this work, combined with the results of our earlier work, show that, while the secondary structures of the wild-type FP and its mutants do not affect the fusogenic activities, the conformational flexibility appears to be an important factor. The active WT FP and its partially active mutants, G3V and G5V, all have significant conformational transitions at one of the glycine sites. They occur at Gly⁵ in FP-wt, at Gly¹⁰ in FP-G5V and at Gly¹³ in FP-G3V. Thus, a glycine site in each of these active (or partially active) FPs provides conformational flexibility. On the other hand, the inactive mutants FP-G10V, FP-L9R and FP-V2E do not have any conformational transitions except at either terminus and thus possess no conformational flexibility. Thus, the results of this work support the suggestion that the role of glycine residues in the fusion domain is to provide the necessary conformational flexibility for fusion activity.The glycines also form a “glycine strip” in the FP that locates on one (the less hydrophobic) face of the helix (the “sided helix”). However, whether this “glycine strip” is disrupted or not does not seem to correlate with the retention of fusogenic activities. Finally, although the FLGFL (8-12) motif is absolutely conserved in the HIV fusion domain, a well-structured motif stabilized by hydrogen bonding does not appear to be required for activity. In fact, hydrogen bonding in this motif was found to be missing in FP-G3V and FP-G5V. Both of these mutants are partially active.     

Analysis of 5' nucleotidase and alkaline phosphatase by gene disruption in Dictyostelium

In Dictyostelium discoideum a phosphatase with a high pH optimum is known to increase in activity during cell differentiation and become localized to a narrow band of cells at the interface of prespore and prestalk cells. However, it was not clear if this activity is due to a classical "alkaline phosphatase" with broad range substrate specificity or to a "5'nucleotidase" with high substrate preference for 5'AMP. We attempted to disrupt the genes encoding these two phosphatase activities in order to determine if the activity that is localized to the interface region resides in either of these two proteins. During aggregation of 5nt null mutants, multiple tips formed rather than the normal single tip for each aggregate. In situ phosphatase activity assays showed that the wt and the 5nt gene disruption clones had normal phosphatase activity in the area between prestalk and prespore cell types, while the alp null mutants did not have activity in this cellular region. Thus, the phosphatase activity that becomes localized to the interface of the prestalk and prespore cells is alkaline phosphatase.     

Activities of anionic and cationic trypsins in the temperature range from 5 to 37 degrees C. Mutant anionic trypsins as a model of cold-adapted (psychrophilic) enzymes

Temperature dependences of kinetic constants (k _cat and K _m) were studied for enzymatic hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-arginine-p-nitroanilide and N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-lysine-p-nitroanilide by bovine cationic and rat anionic (wild-type and mutant) trypsins. The findings were compared with the corresponding literature data for hydrolysis of N-benzoyl-DL-arginine-p-nitroanilide by bovine cationic trypsin and natural trypsins of coldadapted fishes. The anionic and cationic trypsins were found to differ in organization of the S₁ -substrate-binding pocket. The difference in the binding of lysine and arginine residues to this site (S₁) was also displayed by opposite temperature dependences of hydrolysis constants for the corresponding substrates by the anionic and cationic trypsins. The data suggest that the effect of any factor on the binding of substrates (the K _m value) to the anionic and cationic trypsins and on the catalytic activity k _cat should be compared only with the corresponding data for the natural enzyme of the same type. Mutants of rat anionic trypsin at residues K188 or Y228 were prepared by site-directed mutagenesis as approximate models of natural psychrophilic trypsins. Substitution of the charged lysine residue in position 188 by hydrophobic phenylalanine residue shifted the pH optimum of the resulting mutant trypsin K188F from 8.0 to 9.0-10.0, similarly to the case of some natural psychrophilic trypsins, and also 1.5-fold increased its catalytic activity at low temperatures as compared to the wild-type enzyme.     

Contents Volume 93 (1982)

A thymidine kinase heterozygote was isolated from a diploid human lymphoblast line which forms colonies with high efficiency in microtiter dishes. We show that this cell line, called TK6, can be mutated from a tK^+/? to TK^?/? state by diverse mutagens, including ethyl methanesulfonate, butyl methanesulfonate, nitrosomethylurea, UV light, ICR-191, 4-nitroquinoline oxide, fluorodeoxyuridine, benzo[a]pyrene and aflatoxin B₁. We report here the experiments required to demonstrate the applicability of this new line in quantitative assays of mutation in human cells.Mitotic recombination between the centromere and the tk locus could not be induced by either dimethylsulfoxide or phorbol-12-myristate-13-acetate.