In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (＞1013 different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.     

Study on fibroin heavy chain of the silkwormBombyx mori by fluorescencein situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized theFib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified thatFib-H has only one locus, thus providing a temporary solution to the problem about its localization.     

Bioconversion of starch processing waste to Phellinus linteus mycelium in solid-state cultivation

The objective of the experiment was to use starch processing waste as an alternative growth medium for cultivation of mycelia of the mushroom Phellinus linteus and to find an optimum condition under solid-state cultivation. Response surface analysis along with a central composite design was successfully applied to approximate the simultaneous effects of the substrate concentration (16-36 g l(-1)), pH (4.5-6.5), and temperature (25-35 degrees C) on the mycelial growth rate. In the model, pH and temperature significantly affected the mycelial growth but substrate concentration did not. The optimal substrate concentration, pH, and temperature for maximizing growth rate of P. linteus mycelia were found to be 16.5 g l(-1), pH 6.0, and 29.7 degrees C, respectively. Subsequent verification of these levels agreed with model predictions and the maximum mycelial growth rate at these conditions was 6.1 +/- 0.8 mm day(-1). Therefore, the results of the experiments suggest that starch processing waste could be utilized as a growth substrate for the cultivation of the mushroom mycelia of P. linteus, enhancing the usefulness of this byproduct of the starch manufacturing industry. This approach is likely to be useful for establishing similar parameters for the cultivation of other fungi.     

绿洲农业生态经济系统的结构与功能分析以张掖绿洲为例

通过分析张掖绿洲农业生态经济系统的结构与功能,发现其种群结构单一、产业结构不甚合理、空间结构脆弱,决定了绿洲功能较差,产投比低下的特点,并据此从可持续发展的角度提出了改善绿洲农业生态经济系统结构、提高其功能的对策与建议     

In vitro symplasmata formation in the rice diazotrophic endophyte Pantoea agglomerans YS19

Pantoea (formerly Enterobacter) agglomerans YS19 is an endophytic diazotrophic bacterium isolated from rice (Oryza sativa cv. Yuefu) grown in temperate climatic regions in west Beijing (China). The bacterium forms aggregate structures called `symplasmata'. A symplasmatum is a multicellular aggregate structure in which several (at least two) to hundreds of individual cells tightly bind together. The studies on the symplasmata formation of YS19 showed that there were two growth stages for YS19, including the single cell stage existing before exponential growth phase and the symplasmata forming stage starting at the early stationary growth phase in liquid GY (glucose yeast extract) medium or at the end of the exponential growth phase in liquid LB (Luria-Bertani) medium. There was a correlation between symplasmata formation and bacterial growth phase. When the medium was acidified, the cell growth rate was affected by the low pH of the medium, but the time required for symplasmata formation was not influenced by it. YS19 also formed symplasmata on agar medium, where more symplasmata were formed than in liquid medium. The volume of individual constitutional cells of symplasmata was sharply decreased by more than a half in comparison with that of the single cells existing before symplasmata formation. On all the media tested, YS19 formed symplasmata in most of the cell growth phases. The genome DNA/DNA homology between P. agglomerans YS19 and type strain P. agglomerans JCM1236^T (ATCC27155^T) was determined as 90.1%, confirming its membership of P. agglomerans. In order to investigate the phylogenetic relationships of YS19 at the intraspecific, intrageneric and super-generic level, the 16S rDNA similarities between strain YS19 and 17 other strains of Pantoea and 4 representatives of the closely related genera were analyzed. All the strains of Pantoea were clustered into 5 groups, and YS19 was clustered in a unique branch. The 16S rDNA similarity between YS19 and type strain JCM1236^T was 93.9%, much lower than the generally accepted value (=97%) for members of the same species, indicating that the 16S rDNA of YS19 has a distinct molecular characteristic.     

Biosynthesis of rubradirin as an ansamycin antibiotic from Streptomyces achromogenes var. rubradiris NRRL3061

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains. The GeneBank accession number for the sequence reported in this paper is AJ871581.     

Cloning and functional expression of the gene encoding an inhibitor against <Emphasis Type="Italic">Aspergillus flavus</Emphasis> α-amylase,a novel seed lectin from <Emphasis Type="Italic">Lablab purpureus</Emphasis> (<Emphasis Type="Italic">Dolichos lablab</Emphasis>)

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.     

Molecular characterization of the sheep <Emphasis Type="Italic">CIB1</Emphasis> gene

The calcium and integrin binding protein 1(CIB1), is an EF-hand-containing protein that binds many effector proteins including the platelet αIIbβ3 integrin and potentially regulates their functions. Here we report the cloning and characterization of the sheep CIB1 gene. The CIB1 cDNA is 885-bp in size, containing a 45-bp of 5′ untranslated region (UTR), a 264-bp long 3′-UTR and a 576-bp open reading frame that encodes 191 amino acids. The sheep CIB1 cDNA shows 98.3, 92.0, 91.8, 91.3, 90.5 and 90.1% of similarity, at the nucleotide level, to its equivalents in cattle, pigs, rhesus monkey, humans, rats and mice, respectively at the deduced protein level, the corresponding values are more than 94%. The sheep CIB1 gene consisted of seven exons. Quantitative PCR (Q-PCR) showed that CIB1 was widely expressed in different tissues with the highest level in the testis, suggesting that it may play a role in ram fertility. We cloned the sheep CIB2, CIB3 and CIB4 genes and detected their expression patterns in different tissues.     

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.