Genome sequencing and analysis of Aspergillus oryzae

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.     

Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus

Aspergillus fumigatus is exceptional among microorganisms in being both a primary and opportunistic pathogen as well as a major allergen. Its conidia production is prolific, and so human respiratory tract exposure is almost constant. A. fumigatus is isolated from human habitats and vegetable compost heaps. In immunocompromised individuals, the incidence of invasive infection can be as high as 50% and the mortality rate is often about 50% (ref. 2). The interaction of A. fumigatus and other airborne fungi with the immune system is increasingly linked to severe asthma and sinusitis. Although the burden of invasive disease caused by A. fumigatus is substantial, the basic biology of the organism is mostly obscure. Here we show the complete 29.4-megabase genome sequence of the clinical isolate Af293, which consists of eight chromosomes containing 9,926 predicted genes. Microarray analysis revealed temperature-dependent expression of distinct sets of genes, as well as 700 A. fumigatus genes not present or significantly diverged in the closely related sexual species Neosartorya fischeri, many of which may have roles in the pathogenicity phenotype. The Af293 genome sequence provides an unparalleled resource for the future understanding of this remarkable fungus.     

松墨天牛致病菌葡萄曲霉安全性研究

松墨天牛生葡萄曲霉Aspergillus vitis分生孢子对水蚤、果蝇、鲤鱼和对小白鼠经口、经皮、吸入急性毒性试验结果表明：水蚤在20mg／L浓度下72h死亡率23.3％;果蝇在浓度为21mg／L下72h死亡率15．0％;鲤鱼在浓度为22mg／L时72h死亡率4．0％。用3种途径对小鼠的试验表明,小鼠无死亡现象发生,小鼠的体重也无明显影响。该葡萄曲霉对这些实验动物表现为低毒或微毒。     

海洋烟曲霉菌代谢活性物质的萃取

以海洋烟曲霉菌为出发菌,在适宜的发酵条件下进行培养,提取代谢产物并检测其对白色念珠菌抑菌性.分别以硫酸铵、石油醚、乙酸乙酯为萃取剂,研究硫酸铵质量分数和有机溶剂的pH值对海洋烟曲霉菌代谢活性物质萃取效果的影响.结果表明,粗酶液的适宜萃取条件:萃取剂为石油醚,pH值为6,粗酶液与有机溶剂的体积比为1∶1.采用质量分数为70%的硫酸铵沉淀,萃取活性物质的酶活性最大为16.0μkat.L-1,比原粗酶液的酶活(7.9μkat.L-1)提高1倍;而采用石油醚为萃取剂,所萃取活性物质的酶活性最大为20.5μkat.L-1,比原粗酶液的酶活提高2.58倍,且抑菌性能最好,抑菌率达60%左右.     

利用枯草杆菌整合表达嗜铬粒蛋白抗真菌片段

为实现CGA1-76片段基因在枯草杆菌中的稳定表达,将CGA1-76片段基因的表达元件重组到枯草杆菌转座子质粒pHV1249微转座子mini-Tn10内,利用mini-Tn10将CGA1-76片段基因的表达元件整合到枯草杆菌DB1342染色体上,用氯霉素和红霉素筛选枯草杆菌转座工程菌DB1342(TnSVTQ).DB1342(TnSVTQ)在无选择压力条件下经10 d连续传代培养,转座子内氯霉素抗性丢失率为0%,说明转座子稳定整合在DB1342染色体上.SDS-PAGE和免疫印迹分析结果表明,在蔗糖诱导下,CGA1-76片段基因在DB1342(TnSVTQ)中获得表达,产物被分泌到细胞外.孔穴琼脂扩散法测定表达上清的抑制真菌活性,结果表明重组CGA1-76能抑制烟曲霉菌和黄曲霉菌的生长,抑菌圈直径分别为9 mm和8 mm.     

The SH2/SH3 adaptor Grb4 transduces B-ephrin reverse signals

Bidirectional signals mediated by membrane-anchored ephrins and Eph receptor tyrosine kinases have important functions in cell-cell recognition events, including those that occur during axon pathfinding and hindbrain segmentation. The reverse signal that is transduced into B-ephrin-expressing cells is thought to involve tyrosine phosphorylation of the signal's short, conserved carboxy-terminal cytoplasmic domain. The Src-homology-2 (SH2) domain proteins that associate with activated tyrosine-phosphorylated B-subclass ephrins have not been identified, nor has a defined cellular response to reverse signals been described. Here we show that the SH2/SH3 domain adaptor protein Grb4 binds to the cytoplasmic domain of B ephrins in a phosphotyrosine-dependent manner. In response to B-ephrin reverse signalling, cells increase FAK catalytic activity, redistribute paxillin, lose focal adhesions, round up, and disassemble F-actin-containing stress fibres. These cellular responses can be blocked in a dominant-negative fashion by expression of the isolated Grb4 SH2 domain. The Grb4 SH3 domains bind a unique set of other proteins that are implicated in cytoskeletal regulation, including the Cbl-associated protein (CAP/ponsin), the Abl-interacting protein-1 (Abi-1), dynamin, PAK1, hnRNPK and axin. These data provide a biochemical pathway whereby cytoskeletal regulators are recruited to Eph-ephrin bidirectional signalling complexes.     

Controlling Pathogens and Allergens in Composting

IntroductionThe composting process has essentially twoprincipal objectives:.sanitisation—— to reduce significantly the riskfrom pathogens;and.stabilisation—— to produce an “odour free”compost which benefits plant growth.This paper concerns itself with designingcomposting systems to give a productwhich is safeto use from a pathogen point of view.The parallelobjective of achieving stabilisation can be designedto happen concurrently but in most controlledsystems sanitisation happens more ra…     

烟曲霉细胞壁及其GPI锚定结构研究进展

对烟曲霉而言,细胞壁不仅作为其坚硬的外骨骼保证了结构的完整性,对细胞进行保护,而且介导了真菌内部生理状态与外界环境之间的交流。由于缺乏精确快速的诊断方法且抗曲霉感染药物疗效不佳,以及烟曲霉菌株对新药耐受的不断增强,导致真菌感染的死亡率非常高。近年来,以细胞壁为药物靶点的研究越来越多。本文综述了烟曲霉细胞壁的研究进展以及与细胞壁密切相关的GPI锚定结构的研究现状,揭示了抗真菌新药的研制任重道远,迫切需要研究宿主菌的毒力分子机制,寻找更好的药物靶点。     

Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae

The aspergilli comprise a diverse group of filamentous fungi spanning over 200 million years of evolution. Here we report the genome sequence of the model organism Aspergillus nidulans, and a comparative study with Aspergillus fumigatus, a serious human pathogen, and Aspergillus oryzae, used in the production of sake, miso and soy sauce. Our analysis of genome structure provided a quantitative evaluation of forces driving long-term eukaryotic genome evolution. It also led to an experimentally validated model of mating-type locus evolution, suggesting the potential for sexual reproduction in A. fumigatus and A. oryzae. Our analysis of sequence conservation revealed over 5,000 non-coding regions actively conserved across all three species. Within these regions, we identified potential functional elements including a previously uncharacterized TPP riboswitch and motifs suggesting regulation in filamentous fungi by Puf family genes. We further obtained comparative and experimental evidence indicating widespread translational regulation by upstream open reading frames. These results enhance our understanding of these widely studied fungi as well as provide new insight into eukaryotic genome evolution and gene regulation.     

Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair

Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme, or Escherichia coli LigA, an NAD(+)-dependent ligase. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.     

烟曲霉细胞壁作为药物靶标的鉴定研究

烟曲霉(Aspergillus fumigatus)作为侵袭性真菌感染的三大机会病原真菌之一,因其高致病率和高致死率而成为威胁人类健康的重大隐患。有限的抗真菌类药物以及近年来耐药性菌株的涌现使得寻找新的药物靶标并开发新型抗真菌药物迫在眉睫。真菌细胞壁作为真菌所特有的结构一直以来被认为是理想的药物靶标。本文概述了烟曲霉细胞壁结构及合成过程的相关研究进展,并分析了参与细胞壁各组分合成的酶作为药靶的可行性。     

Structure and conserved RNA binding of the PAZ domain

The discovery of RNA-mediated gene-silencing pathways, including RNA interference, highlights a fundamental role of short RNAs in eukaryotic gene regulation and antiviral defence. Members of the Dicer and Argonaute protein families are essential components of these RNA-silencing pathways. Notably, these two families possess an evolutionarily conserved PAZ (Piwi/Argonaute/Zwille) domain whose biochemical function is unknown. Here we report the nuclear magnetic resonance solution structure of the PAZ domain from Drosophila melanogaster Argonaute 1 (Ago1). The structure consists of a left-handed, six-stranded beta-barrel capped at one end by two alpha-helices and wrapped on one side by a distinctive appendage, which comprises a long beta-hairpin and a short alpha-helix. Using structural and biochemical analyses, we demonstrate that the PAZ domain binds a 5-nucleotide RNA with 1:1 stoichiometry. We map the RNA-binding surface to the open face of the beta-barrel, which contains amino acids conserved within the PAZ domain family, and we define the 5'-to-3' orientation of single-stranded RNA bound within that site. Furthermore, we show that PAZ domains from different human Argonaute proteins also bind RNA, establishing a conserved function for this domain.     

APOBEC3 inhibits mouse mammary tumour virus replication in vivo

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3(-/-) mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.     

Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth.

Neovascularization is critical for the growth of tumours and is a dominant feature in a variety of angiogenic diseases such as diabetic retinopathy, haemangiomas, arthritis and psoriasis. Recognition of the potential therapeutic benefit of controlling unabated capillary growth has led to a search for safe and effective angiogenesis inhibitors. We report here the synthesis of a family of novel inhibitors that are analogues of fumagillin, a naturally secreted antibiotic of Aspergillus fumigatus fresenius. We first isolated this fungus from a contaminated culture of capillary endothelial cells. Purified fumagillin inhibited endothelial cell proliferation in vitro and tumour-induced angiogenesis in vivo; it also inhibited tumour growth in mice, but prolonged administration was limited because it caused severe weight loss. Synthesis of fumagillin analogues yielded potent angiogenesis inhibitors ('angioinhibins') which suppress the growth of a wide variety of tumours with relatively few side-effects.     

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.     

高效降解秸秆纤维素菌株的筛选鉴定及产酶条件优化

从堆沤秸秆土壤中筛选到一株高产纤维素酶菌株YA-14,经菌株形态特性分析和18SrRNA基因序列分析,确定其为烟曲霉(Aspergillus Fumigatus).通过单因素及正交试验,研究了多种因素对菌株产酶效果的影响,确定最佳产酶条件为碳氮比2:1,初始pH值为5,Mg2+ 浓度为0.4 mg/mL,接种量为1.0...     

竹材真菌的研究

对发霉变色竹材上的真菌进行了分离培养和鉴定。结果表明用组织分离得到了３个真菌分离物；用孢子分离得到了６个真菌分离物；而用子实体直接鉴定的有４个种。它们隶属于半知菌亚门的两个纲，３个目，１１个属的真菌。在竹材上最常见的真菌是葡萄生交链孢（Ａｌｔｅｒｎａｒｉａｖｉｔｉｃｏｌａ），局限曲霉（Ａｓｐｅｒｇｉｌｌｕｓｒｅｓｔｒｉｃｔｕｓ），多主枝孢霉（Ｃｌａｄｏｓｐｏｒｉｕｍｈｅｒｂａｒｕｍ）和青霉（Ｐｅｎｉｃｉｌｌｉｕｍｓｐ．）。     

竹醋液抗真菌的体外敏感性对比试验研究

基于沙保弱氏培养基,采用试管法和打孔法测定了竹醋液对6种致病真菌的最低抑菌浓度、最低杀菌浓度和抑菌圈直径.以常用市售抗真菌药作为对照,证实了竹醋液在体外有较显著的抗真菌和杀菌作用.     

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.