Solution structure of the severe acute respiratory syndrome-coronavirus heptad repeat 2 domain in the prefusion state

The envelope glycoprotein, termed the spike protein, of severe acute respiratory syndrome coronavirus (SARS-CoV) is known to mediate viral entry. Similar to other class 1 viral fusion proteins, the heptad repeat regions of SARS-CoV spike are thought to undergo conformational changes from a prefusion form to a subsequent post-fusion form that enables fusion of the viral and host membranes. Recently, the structure of a post-fusion form of SARS-CoV spike, which consists of isolated domains of heptad repeats 1 and 2 (HR1 and HR2), has been determined by x-ray crystallography. To date there is no structural information for the prefusion conformations of SARS-CoV HR1 and HR2. In this work we present the NMR structure of the HR2 domain (residues 1141-1193) from SARS-CoV (termed S2-HR2) in the presence of the co-solvent trifluoroethanol. We find that in the absence of HR1, S2-HR2 forms a coiled coil symmetric trimer with a complex molecular mass of 18 kDa. The S2-HR2 structure, which is the first example of the prefusion form of coronavirus envelope, supports the current model of viral membrane fusion and gives insight into the design of structure-based antagonists of SARS.     

Activity of the 5′ regulatory regions of the rice polyubiquitin <Emphasis Type="Italic">rubi3</Emphasis> gene in transgenic rice plants as analyzed by both <Emphasis Type="Italic">GUS</Emphasis> and <Emphasis Type="Italic">GFP</Emphasis> reporter genes

Ubiquitin is an abundant protein involved in protein degradation and cell cycle control in plants and rubi3 is a polyubiquitin gene isolated from rice (Oryza sativa L.). Using both GFP and GUS as reporter genes, we analyzed the expression pattern of the rubi3 promoter as well as the effects of the rubi3 5'-UTR (5' untranslated region) intron and the 5' terminal 27 bp of the rubi3 coding sequence on the activity of the promoter in transgenic rice plants. The rubi3 promoter with the 5'-UTR intron was active in all the tissue and cell types examined and supported more constitutive expression of reporter genes than the maize Ubi-1 promoter. The rubi3 5'-UTR intron mediated enhancement on the activity of its promoter in a tissue-specific manner but did not alter its overall expression pattern. The enhancement was particularly intense in roots, pollen grains, inner tissue of ovaries, and embryos and aleurone layers in maturing seeds. The translational fusion of the first 27 bp of the rubi3 coding sequence to GUS gene further enhanced GUS expression directed by the rubi3 promoter in all the tissues examined. The rubi3 promoter should be an important addition to the arsenal of strong and constitutive promoters for monocot transformation and biotechnology.     

球形芽孢杆菌的抗药标记及外源DNA的转化与表达

用亚硝基胍（NTG）对球形芽孢杆菌（Bacillussphaericus）进行化学诱变,筛选到利福平（Rif）和链霉素（Sm）二个标记菌株。抗药浓度均达100u／ml培养基。其抗药性状能够获得较好地遗传。用含溶葡球菌酶基因的质粒DNA对RifR菌株进行原生质体转化,酶基因在该抗药菌株中获得了高效表达。经摇瓶发酵试验,溶葡球菌酶的活性约为122u／ml培养液。     

Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in pseudomonas aeruginosa

The complete genome of the bacterial pathogen Pseudomonas aeruginosa has now been sequenced, allowing gene deletion, one of the most frequently used methods in gene function study, to be fully exploited. In this study, we combine the sacB-based negative selection system with a cre-lox antibiotic marker recycling method. This methodology allows allelic exchange between a target gene and a gentamicin cassette flanked by the two lox sequences. A tetracycline plasmid expressing the cre recombinase is then introduced in the mutant strain to catalyze the excision of the lox-flanked resistance marker. We demonstrate here the efficiency of the combination of these two methods in P. aeruginosa by successively deleting ExoS and ExoT, which are two genetically independent toxins of the type-three secretion system (TTSS). This functional cre-lox recycling antibiotic marker system can create P. aeruginosa strains with multiple mutations without modifying the antibiotic resistance profile when compared to the parental strain.     

蜜蜂TPI基因克隆与生物信息学预测

利用电子克隆方法获得蜜蜂(Apis mellifera)磷酸甘油醛异构酶(triosephosphate isomerase,TPI)基因,并采用生物信息学方法对该基因编码蛋白从等电点、疏水性/亲水性、二级结构等进行了预测,以及试验验证,结果表明蜜蜂TPI基因全长为1 768 bp,具有完整的开放阅读框架(ORF),并得到了试验证实.     

黄瓜钝绥螨对茶黄螨雌成螨和腐食酪卵的功能反应

研究黄瓜钝绥螨Amblyseius cucumeris 对茶黄螨Polyphagotarsonemus latus (Banks)雌成螨和腐食酪螨Tyrophagus putrescentiae卵的功能反应。结果表明,黄瓜钝绥螨的第1若螨,第2若螨,雌成螨捕食茶黄螨雌成螨和腐食酪螨卵的功能反应均属于Holling II型,其中,雌成螨的捕食能力最强,对腐食酪螨卵和对茶黄螨雌成螨的攻击系数a大,处理时间th短,第2若螨也具有较强的捕食能力,对静态的腐食酪螨卵比对动态的茶黄螨捕食能力强,黄瓜钝绥螨对茶黄螨雌成螨具有很强的捕食能力。     

Activated PAK4 regulates cell adhesion and anchorage-independent growth

The serine/threonine kinase PAK4 is an effector molecule for the Rho GTPase Cdc42. PAK4 differs from other members of the PAK family in both sequence and function. Previously we have shown that an important function of this kinase is to mediate the induction of filopodia in response to activated Cdc42. Since previous characterization of PAK4 was carried out only with the wild-type kinase, we have generated a constitutively active mutant of the kinase to determine whether it has other functions. Expression of activated PAK4 in fibroblasts led to a transient induction of filopodia, which is consistent with its role as an effector for Cdc42. In addition, use of the activated mutant revealed a number of other important functions of this kinase that were not revealed by studying the wild-type kinase. For example, activated PAK4 led to the dissolution of stress fibers and loss of focal adhesions. Consequently, cells expressing activated PAK4 had a defect in cell spreading onto fibronectin-coated surfaces. Most importantly, fibroblasts expressing activated PAK4 had a morphology that was characteristic of oncogenic transformation. These cells were anchorage independent and formed colonies in soft agar, similar to what has been observed previously in cells expressing activated Cdc42. Consistent with this, dominant-negative PAK4 mutants inhibited focus formation by oncogenic Dbl, an exchange factor for Rho family GTPases. These results provide the first demonstration that a PAK family member can transform cells and indicate that PAK4 may play an essential role in oncogenic transformation by the GTPases. We propose that the morphological changes and changes in cell adhesion induced by PAK4 may play a direct role in oncogenic transformation by Rho family GTPases and their exchange factors.     

Genetic and physiological studies of Bacillus subtilis sigma A mutants defective in promoter melting.

The Bacillus subtilis sigA gene encodes the primary sigma factor of RNA polymerase and is essential for cell growth. We have mutated conserved region 2.3 of the sigma A protein to substitute each of seven aromatic amino acids with alanine. Several of these aromatic amino acids are proposed to form a melting motif which facilitates the strand separation step of initiation. Holoenzymes containing mutant sigma factors recognize promoters, but some are defective for DNA melting in vitro. We have studied the ability of each mutant sigma factor to support cell growth by gene replacement and complementation. The two region 2.3 mutants least impaired in promoter melting in vitro (Y180A and Y184A) support cell growth in single copy, although the Y184A allele imparts a slow-growth phenotype at low temperatures. A strain expressing only the Y189A variant of the sigma A protein, known to be defective in DNA melting in vitro, grows very slowly and is altered in its pattern of protein synthesis. Only the wild-type and Y180A sigma A proteins efficiently complement a temperature-sensitive allele of sigA. Overexpression of three of the sigma A proteins defective for promoter melting in vitro (Y189A, W192A, and W193A) leads to a decrease in RNA synthesis and cell death. These results indicate that mutations which specifically impair DNA melting in vitro also impair sigma function in vivo and therefore support the hypothesis that sigma plays an essential role in both DNA melting and promoter recognition.     

造纸废水微生物絮凝剂菌株的分离鉴定及特性

从造纸废水处理厂卡鲁赛尔(Carrousel)氧化沟的活性污泥样品中分离到了1株产絮凝剂的菌株B-6,经生理生化试验和16Sr DN A基因序列分析,鉴定为芽胞杆菌属(Bacillus sp.)。该菌株产生的絮凝剂具有良好的酸碱稳定性,在pH值1～5和7～11范围内,其絮凝活性维持在80%以上。优化该菌发酵上清液的絮凝条件,结果表明,加入5 mL的1%CaCl2,发酵上清液投加量为0.8 mL时,该菌株发酵上清液对高岭土悬浊液的絮凝率可达95.4%。