Protein conformational dynamics probed by single-molecule electron transfer

Electron transfer is used as a probe for angstrom-scale structural changes in single protein molecules. In a flavin reductase, the fluorescence of flavin is quenched by a nearby tyrosine residue by means of photo-induced electron transfer. By probing the fluorescence lifetime of the single flavin on a photon-by-photon basis, we were able to observe the variation of flavin-tyrosine distance over time. We could then determine the potential of mean force between the flavin and the tyrosine, and a correlation analysis revealed conformational fluctuation at multiple time scales spanning from hundreds of microseconds to seconds. This phenomenon suggests the existence of multiple interconverting conformers related to the fluctuating catalytic reactivity.     

Disulfide isomerization after membrane release of its SAR domain activates P1 lysozyme

The P1 lysozyme Lyz is secreted to the periplasm of Escherichia coli and accumulates in an inactive membrane-tethered form. Genetic and biochemical experiments show that, when released from the bilayer, Lyz is activated by an intramolecular thiol-disulfide isomerization, which requires a cysteine in its N-terminal SAR (signal-arrest-release) domain. Crystal structures confirm the alternative disulfide linkages in the two forms of Lyz and reveal dramatic conformational differences in the catalytic domain. Thus, the exported P1 endolysin is kept inactive by three levels of control-topological, conformational, and covalent-until its release from the membrane is triggered by the P1 holin.     

5株拮抗细菌对链孢霉的拮抗作用

用平板对峙法筛选出5株对链胞霉有拮抗作用的细菌。结果表明,SK-12、SK-10对链孢霉的拮抗能力最强;浓缩后拮抗细菌拮抗能力显著提高;浓度为20%时,SK-1、SK-10、SK-11对W-N、R-N的抑菌率均达到100%,且培养6d后,W-N仍然不能生长。     

Mutagenic and recombinogenic activities of the food additive furylfuramide in eurkaryotes

The mutagenic and recombinogenic activities of furylfuramide, an antimicrobial food additive, were tested in a two-component heterokaryon of Neurospora crassa and a diploid strain of Saccharomyces cerevisiae, respectively. The results show that furylfuramide is genetically active in both eukaryotic organisms; it induces mutations in Neurospora crassa and mitotic crossing over in Saccharomyces cerevisiae.     

Heterogeneity of 5S RNA in fungal ribosomes

Neurospora crassa has at least seven types of 5S RNA genes (alpha, beta, gamma, epsilon, delta, zeta, and eta) with different coding regions. A high resolution gel electrophoresis system was developed to separate minor 5S RNA's from the major 5S RNA (alpha). A study of several Neurospora crassa strains, four other species in the genus Neurospora, members of two closely related genera, and three distantly related genera demonstrated that 5S RNA heterogeneity is common among fungi. In addition, different 5S RNA's are present in Neurospora ribosomes. The finding that fungal ribosomes are structurally heterogeneous suggests that ribosomes may be functionally heterogeneous as well.     

Photoejection of electrons from flavins in polar media

Riboflavin and 12 of its derivatives have been shown to form solvated electrons under ultraviolet irradiation (253.7 nanometers) in various water-methanol solvent mixtures. The highest quantum yield of solvated electrons (about 0.03) was obtained for flavins containing tyrosine on a side chain in the isoalloxazine N-3 or N-10 position. The splitting of hydrogen atoms from excited flavin molecules was also observed. From the results presented here, it can be determined that the semiquinone transients are formed not only by way of the flavin triplet, as usually suggested, but also by the attack of the electrons and hydrogen atoms on flavin molecules in the ground state. This is important, because the flavin radicals remaining after the electron-ejection or hydrogen-splitting processes must also be considered in the subsequent reaction mechanisms. The electron-ejection process from electronically excited flavins has important implications in the photobiology of these compounds.     

DNAs of the two mating-type alleles of Neurospora crassa are highly dissimilar

The mating-type alleles A and a of Neurospora crassa control mating in the sexual cycle and function in establishing heterokaryon incompatibility in the vegetative cycle. The A and a alleles were cloned, and they were shown to encode both the sexual functions and vegetative incompatibility. The mating-type clones contain nonhomologous DNA segments that are flanked by common DNA sequences. Neurospora crassa and all heterothallic and pseudohomothallic Neurospora species contain a single copy of one mating-type sequence or the other within each haploid genome. The six known self-fertile homothallic isolates contain an A homolog, but only one species also contains a homologous sequences. Homothallism in these species is not due to mating-type switching, as it is in Saccharomyces cerevisiae.     

The Neurospora checkpoint kinase 2: a regulatory link between the circadian and cell cycles

The clock gene period-4 (prd-4) in Neurospora was identified by a single allele displaying shortened circadian period and altered temperature compensation. Positional cloning followed by functional tests show that PRD-4 is an ortholog of mammalian checkpoint kinase 2 (Chk2). Expression of prd-4 is regulated by the circadian clock and, reciprocally, PRD-4 physically interacts with the clock component FRQ, promoting its phosphorylation. DNA-damaging agents can reset the clock in a manner that depends on time of day, and this resetting is dependent on PRD-4. Thus, prd-4, the Neurospora Chk2, identifies a molecular link that feeds back conditionally from circadian output to input and the cell cycle.     

基于大学生素质教育的视听服务及视听文献建设策略

形象生动、直观性强的视听文献在大学生素质教育中的优势显而易见。近年来,多媒体技术、数字应用技术的发展,视听资源比例明显增多,视听文献越来越受到读者尤其是大学生读者的喜爱。因此要加强视听文献的建设与发展,为培养高素质的复合型人才发挥更大的作用。     

A portable signal causing faithful DNA methylation de novo in Neurospora crassa

Methylation of cytosine residues in eukaryotic DNA is common, but poorly understood. Typically several percent of the cytosines are methylated; however, it is unclear what governs which sequences eventually become modified. Neurospora crassa DNA containing the "zeta-eta" (zeta-eta) region, which is a region of unusually heavy methylation, was tested for its ability to direct DNA methylation de novo. DNA stripped of its methylation by propagation in Escherichia coli was reintroduced into Neurospora crassa by transformation. The zeta-eta region reproducibly became "properly" methylated whether inserted at its native chromosomal position or at ectopic sites. Adjacent Neurospora and bacterial sequences in the transforming DNA rarely became methylated. A model is presented that accounts for position-independent faithful methylation as observed in the zeta-eta region, as well as position-dependent methylation, as occasionally observed, especially with sequences not native to Neurospora.     

Colonial growth of Neurospora. Sorbose and enzymes alter the composition of the cell wall and induce morphological changes

L-Sorbose, an agent which induces colonial growth in Neurospora crassa, also induces structural changes in the cell wall. Acid hydrolyzates of cell walls isolated from sorbose-grown (colonial) hyphae contain more glucosamine and less glucose than do hydrolyzates of cell walls obtained from normally growing hyphae. Snail digestive juice, an agent which effects a structural change in the cell wall of N. crassa by liberating from it large quantities of glucose, has been found to induce colonial growth.     

Ethylene formation from ethyl moiety of ethionine

In plants, ethylene is formed in the presence of light and flavin mononucleotide from ethionine and S-ethylcysteine. The ethylene is formed from the ethyl moiety of ethionine.     

An essential role of N-terminal arginylation in cardiovascular development

The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues-aspartate, glutamate, and cysteine-are substrates for arginylation. The mouse ATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found that ATE1-/- embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor.     

Efficient method for selection of auxotrophic mutants of Neurospora

A technique for the selection of induced and spontaneous auxotrophic mutants of Neurospora in relatively high frequency is described. The method takes advantage of the reduced rate of death of germinating conidia of an inositol-requiring strain when a secondary mutation is imposed.     

Arginine and pyrimidine biogenesis in Neurospora

The growth-promoting activity of propionic acid and related compounds for the pyrimidine-less strain, Neurospora crassa 1298, is markedly inhibited by arginine. The data suggest that arginine exerts an inhibitory effect upon or represses the synthesis of an enzyme involved in pyrimidine formation.     

Phospholipase-induced crystallization of channels in mitochondrial outer membranes

When outer membranes from Neurospora crassa mitochondria are treated with low levels of phospholipase A2 under continuous dialysis, two-dimensional crystalline arrays of the pore protein component of these membranes are formed.