Mutational tests of the NMR-docked structure of the staphylococcal nuclease–metal–3′,5′-pdTp complex

In the X-ray structure of the staphylococcal nuclease–Ca²⁺ ?3′,5′-pdTp complex, the conformation of the inhibitor 3′,5′-pdTp is distroteed Lys-70* and Lys-71* from an adjacent molecule of staphylococcal nuclease (Loll, P.J., Lattman, E.E. Proteins 5 : 183-201, 1989). In order to correct this crystal packing problem, the solution conformation of enzyme-bound 3′,5′-pdTp in the staphylococcal nuclease–metal–pdTp Complex determined by NMR methods was docked into the X-ray structure of the enzyme [Weber, D. J., Serpersu, E. H., Gittis, A. G., Lattman, E. E., Mildvan, A. S. (preceding paper)]. In the NMR-docked structure, the 5′-phophate of 3′,5′-pdTp overlaps with that in the X-ray Structure. However the 3′-phosphate accepts a hydrogen bond from Lys-49 (2.89Å) rather than from Lys-84 (8.63 Å), and N3 of thymine donates a hydrogen bond to the OH of Tyr-115 (3.16 Å) which does not occur in the X-ray structure (5.28 Å). These interactions have been tested by binding studies of 3′,5′-pdTp, Ca²⁺, and Mn²⁺ to the K49A, K84A, and Y115A mutants of staphylococcal nuclease using water proton relaxation rate and EPR methods. Each mutant was fully active and structurally intact, as found by CD and two-dimensional NMR spectroscopy, but bound Ca²⁺ 9.1- to 9.9-fold more weakly than the wild-type enzyme. While thye K84A mutation did not significantly weaken 3′,5′-pdTp binding to the enzyme (1.5 ± 0.7 fold), the K49A mutation weakened 3′,5′-pdTp binding to the enzyme by the factor of 4.4 ± 1.8-fold. Similarly, the Y115A mutation weakened 3′,5′-pdTp binding to the enzyme 3.6 ± 1.6-fold. Comparable weakening effects of these mutations were found on the binding of Ca²⁺-3′,5′-pdTp. These results are more readily explained by the NMR-docked structure of staphylococcal nuclease-metal-3′,5′-pdTp than by the X-ray structure. © 1993 Wiley-Liss, Inc.     

Specificity of rabbit antisera against the rough lipopolysaccharide of Salmonella minnesota strain R7 (chemotype Rd1P−)

Abstract Rabbit polyclonal antibodies against the lipopolysaccharide (LPS) of the Rd₁P⁻ mutant strain R7 of Salmonella minnesota were serologically characterized using R7 LPS, dephosphorylated LPS, deacylated LPS, deacylated, dephosphorylated and reduced LPS, and synthetic partial structures. The latter comprised partial structures of the core region of Rd₁P⁻ LPS bound to the β 1 → 6-linked glucosamine disaccharide with two amide-linked 3-hydroxytetradecanoic acid residues or artificial glycoconjugates comprised of the synthetic oligosaccharides coupled to bovine serum albumin. Using a passive hemolysis and an enzyme immunoassay, absorption and inhibition experiments, the antibody specificities present could be determined. One group of antibodies required components of the core region and the phosphorylated glucosamine disaccharide of the lipid A moiety for binding. The majority of phosphate-independent antibodies was directed against the trisaccharide l -glycero-α- d -manno-heptopyranose(1 → 3)- l -glycero-α- d -manno-heptopyranose(1 → 5)3-deoxy- d -manno-octulosonic acid. Antibodies against the 1 → 3- and 1 → 7-linked heptose disaccharides and against a single heptose were also detected, however, with low titers. No antibodies were found which required the presence of fatty acids.     

猪链球菌2型Orf207基因缺失菌株的构建及其生物学特性检测

【背景】猪链球菌2型(streptococcus suis type 2, SS2)可引起人、猪的脑膜炎、关节炎及败血症等,不仅给养猪业带来巨大的经济损失,同时严重威胁公共卫生安全。本团队前期通过噬菌体展示文库技术发现Orf207编码蛋白可能参与SS2诱导的脑膜炎发生,然而其在SS2致病过程中的具体作用尚不清楚。【目的】探究Orf207基因对SS2致病性的影响。【方法】采用温敏性自杀质粒介导的同源重组系统,构建SC19 Orf207基因缺失菌株ΔOrf207及其回补菌株CΔOrf207,系统比较缺失菌株与野生株间在生长特性、形态、组织定殖能力、毒力情况、细胞黏附与侵袭及抗巨噬细胞吞噬能力等生物学特性方面的差异。【结果】与野生株相比,缺失菌株链长变短,生长速度略慢;而且Orf207缺失显著增加了小鼠的存活率,降低了细菌在血液、心脏、肝脏、脾脏、肺脏、肾脏、脑组织的定殖能力和对肺组织的病理损伤并显著减弱SS2对HeLa细胞的黏附与侵袭能力及抗巨噬细胞吞噬能力。【结论】Orf207基因可以显著降低SS2对宿主的致病能力,本研究结果不仅丰富了SS2的致病机制,也为SS2疫苗等研发提供了新靶点。     

不同光质对辣椒叶色黄化突变体光合生理特性的影响研究

以辣椒叶色黄化突变体yl1及其野生型6421为试材,用白光、蓝光、红光、绿光、紫光、黄光和远红光不同光质进行处理,考察其表型、生理及光合特性的变化特征,探究光质对黄叶辣椒植株生长发育的影响。结果显示：(1)蓝光与红光对辣椒幼苗的生长有促进作用,黄光和远红光则会显著抑制幼苗生长,6421生长受不同光质的抑制影响比yl1更大。(2)两个辣椒材料光合色素含量在不同光质下均不同程度降低;6421叶绿素总含量和类胡萝卜素含量在不同光质下均高于yl1,yl1和6421叶片的光合色素含量分别在紫光和黄光下最低。(3)蓝光和绿光能显著提高yl1的净光合速率(P_n),而不同光质处理均显著降低了6421的P_n。(4)紫光处理使yl1的PSⅡ潜在活性(F_v/F_o)和最大光化学效率(F_v/F_m)值均显著降低且显著低于6421,但升高了光化学猝灭系数(qP)和非光化学猝灭系数(NPQ)。(5)蓝光、红光和绿光均能提高辣椒的超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(...     

DEVELOPMENTAL MUTANTS OF VOLVOX: DOES MUTATION RECREATE THE PATTERNS OF PHYLOGENETIC DIVERSITY?

The nature of the variation which is created by mutation can show how the direction of evolution is constrained by internal biases arising from development and pre-existing design. We have attempted to quantify these biases by measuring eight life history characters in developmental mutants of Volvox carteri. Most of the mutants in our sample were inferior to the wild type, but deviated by less than tenfold from the wild-type mean. Characters differed in mutability, suggesting different levels of canalisation. Most correlations between life history characters among strains were positive, but there was a significant negative correlation between the size and the number of reproductive cells, suggesting an upper limit to the total quantity of germ produced by individuals. The most extreme phenotypes in our sample were very vigorous, showing that not all mutations of large effect are unconditionally deleterious. We investigated the effect of developmental constraints on the course of evolution by comparing the variance and covariance patterns among mutant strains with those among species in the family Volvocaceae. A close correspondence between patterns at these two levels would suggest that pre-existing design has a strong influence on evolution, while little or no correspondence shows the action of selection. The variance generated by mutation was equal to that generated by speciation in the family Volvocaceae, the genus Volvox, or the section Merillosphaera, depending on the character considered. We found that mutation changes the volume of somatic tissue independently of the quantity of germ tissue, so that the interspecific correlation between soma and germ can be attributed to selection. The negative correlation between size and number of germ cells among mutants of V. carteri is also seen among the larger members of the family (Volvox spp.), but not among the smaller members, suggesting a powerful design constraint that may be responsible for the absence of larger forms in the entire group.     

Lignin genetic engineering

Although lignins play important roles in plants, they often represent an obstacle to the utilization of plant biomass in different areas: pulp industry, forage digestibility. The recent characterization of different lignification genes has stimulated research programmes aimed at modifying the lignin profiles of plants through genetic engineering (antisense and sense suppression of gene expression). The first transgenic plants with a modification of monomeric composition of lignins and lignin content have been recently obtained. Down regulation of the OMT gene induces dramatic reduction of syringyl units. CAD down regulated plants exhibit a unusual red phenotype associated with the developing xylem and several chemical modifications of their lignins including an increase in cinnamaldehydes in the polymer structure. Interestingly this novel lignin is removed more easily during the pulping process. In both OMT and CAD down regulated plants no changes in phenotypic characteristics such as growth architecture and morphology were observed. More recent experiments have shown that a reduction of CCR activity determines specific changes in the coloration of the xylem area suggesting significant chemical modifications which are currently being studied.These different results show that it is possible to manipulate lignins through targeted genetic transformation of plants and that lignins exhibit a relative flexibility of their chemical structure. Future developments should probe the impact of down regulating the expression of other recently characterized lignification genes such as F5H and CCoAOMT and also of a combination of genes in order to tailor lignins more adapted to specific purposes. In addition to biotechnological applications which should provide important economical benefits for utilization of wood in the pulp industry, genetic engineering of lignins offer very promising perspectives for the understanding of lignin synthesis, structure and properties.     

Structural and immunochemical studies on the lipopolysaccharide of the ‘T-antigen’-containing mutant Proteus mirabilis R14/1959

Abstract In DOC-PAGE, lipopolysaccharide (LPS) of Proteus mirabilis R14/1959 (Rb-type) mutant showed a ladder-like migration pattern indicating the presence of a high molecular weight polysaccharide chain. The isolated polysaccharide, called T-antigen because of similarity with the T1 chain of Salmonella friedenau LPS, contained d -glucose, d -galacturonic acid ( d -GalA), and d -GlcNAc in molar ratios 2:1:1 and was structurally different from the O-antigen of the parental S-strain P. mirabilis S1959 but identical to the O-antigen of another S-strain Proteus penneri 42. The importance of a d -GalA( l -Lys)-containing epitope, most likely present in the core region of LPS, and of GalA present in the T-antigen chain in manifesting the serological specificity of P. mirabilis R14/1959 were revealed using rabbit polyclonal homologous and heterologous R- and O-specific antisera and the appropriate antigens, including synthetic antigens which represent partial structures of various Proteus LPS.     

Perturbations of the denatured state ensemble: modeling their effects on protein stability and folding kinetics.

By considering the denatured state of a protein as an ensemble of conformations with varying numbers of sequence-specific interactions, the effects on stability, folding kinetics, and aggregation of perturbing these interactions can be predicted from changes in the molecular partition function. From general considerations, the following conclusions are drawn: (1) A perturbation that enhances a native interaction in denatured state conformations always increases the stability of the native state. (2) A perturbation that promotes a non-native interaction in the denatured state always decreases the stability of the native state. (3) A change in the denatured state ensemble can alter the kinetics of aggregation and folding. (4) The loss (or increase) in stability accompanying two mutations, each of which lowers (or raises) the free energy of the denatured state, will be less than the sum of the effects of the single mutations, except in cases where both mutations affect the same set of partially folded conformations. By modeling the denatured state as the ensemble of all non-native conformations of hydrophobic-polar (HP) chains configured on a square lattice, it can be shown that the stabilization obtained from enhancement of native interactions derives in large measure from the avoidance of non-native interactions in the D state. In addition, the kinetic effects of fixing single native contacts in the denatured state or imposing linear gradients in the HH contact probabilities are found, for some sequences, to significantly enhance the efficiency of folding by a simple hydrophobic zippering algorithm. Again, the dominant mechanism appears to be avoidance of non-native interactions. These results suggest stabilization of native interactions and imposition of gradients in the stability of local structure are two plausible mechanisms involving the denatured state that could play a role in the evolution of protein folding and stability.     

Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses ofPhycomyces blakesleeanus

Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages. Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors.     

Endogenous gibberellin levels influence in-vitro shoot regeneration in Arabidopsis thaliana (L.) Heynh.

The regeneration of shoot buds from callus cells in vitro is an important technique in modern plant genetic manipulation. Whilst it is clear that genetic factors play a major role in determining the ability of callus cells to become organized into regenerating shoot buds, the precise nature of these factors remains unknown. Here we show that callus derived from mutants of Arabidopsis thaliana which have reduced levels of endogenous bioactive gibberellins (GAs), or reduced responsivity to GAs, regenerates shoot buds more readily than does callus derived from wild-type controls. In addition, exogenous GA reduces, and exogenous paclobutrazol (a GA-biosynthesis inhibitor) increases, the frequency of shoot bud regeneration from wild-type callus. These results show that GA levels play a role in regulating shoot bud regeneration from callus, and suggest that variation in endogenous GA levels or responsivity may account for a major component of the genetic variation in shoot bud regeneration frequency described in other species.