Abortive ligation intermediate blocks seamless repair of double-stranded breaks.

Because indel results in frame-shift mutations, seamless repair of double-stranded break (DSB)s plays a pivotal role in synthetic biology, molecular biology, and genome integrity. However, DSB repair is not well documented. T4 DNA ligase (T4lig) served to ligate intra-molecularly a zero bp break-apart DSB linear plasmid DNA pET22b(28a)-xylanase. An ATP T4lig ligation reaction joined one single-stranded break (SSB) into a phosphodiester-bond, whereas the opposite SSB into an abortive ligation intermediate blocking the DSB sequential repair. The intermediate proved to be fluorescent Cy5-AMP-SSB by a T4lig ligation reaction in the aid of Alexa Fluor 647 ATP having Cy5-AMP fluorescence. The fluorescent Cy5-AMP-SSB was de-adenylated into SSB by an ATP-free T4lig or Mg2+-free T4ligL159L reaction. The de-adenylated SSB was re-joined into another phosphodiester-bond by a sequential ATP T4lig re-ligation reaction. Thereby, DSB repair proceeds an abortive ligation, a reverse de-adenylation, and a sequential re-ligation reaction. The result has a potential usage in synthetic biology, molecular biology, and cancer-curing.

Phylogenesis of the Functional 1-Aminocyclopropane-1-Carboxylate Oxidase of Fungi and Plants.

The 1-aminocyclopropane-1-carboxylic acid (ACC) pathway that synthesizes ethylene is shared in seed plants, fungi and probably other organisms. However, the evolutionary relationship of the key enzyme ACC oxidase (ACO) in the pathway among organisms remains unknown. Herein, we cloned, expressed and characterized five ACOs from the straw mushroom (Volvariella volvacea) and the oyster mushroom (Pleurotus ostreatus): VvACO1-4 and PoACO. The five mushroom ACOs and the previously identified AbACO of the button mushroom contained all three conserved residues that bound to Fe(II) in plant ACOs. They also had variable residues that were conserved and bound to ascorbate and bicarbonate in plant ACOs and harbored only 1-2 of the five conserved ACO motifs in plant ACOs. Particularly, VvACO2 and AbACO had only one ACO motif 2. Additionally, VvACO4 shared 44.23% sequence identity with the cyanobacterium Hapalosiphon putative functional ACO. Phylogenetic analysis showed that the functional ACOs of monocotyledonous and dicotyledonous plants co-occurred in Type I, Type II and Type III, while putative functional gymnosperm ACOs also appeared in Type III. The putative functional bacterial ACO, functional fungi and slime mold ACOs were clustered in ancestral Type IV. These results indicate that ACO motif 2, ACC and Fe(II) are essential for ACO activity. The ACOs of the other organisms may come from the horizontal transfer of fungal ACOs, which were found ordinarily in basidiomycetes. It is mostly the first case for the horizontal gene transfers from fungi to seed plants. The horizontal transfer of ACOs from fungi to plants probably facilitates the fungal-plant symbioses, plant-land colonization and further evolution to form seeds.

Mitochondrial genome characterization and phylogenetic analysis of arbuscular mycorrhizal fungus Rhizophagus sp.

In the present study, the complete mitochondrial genome of Rhizophagus sp. was assembled by the next-generation sequencing. We found that the complete mitochondrial genome of Rhizophagus sp. is 50,449 bp in length and consists of 14,741 (29.22%) adenine, 9427 (18.69%) cytosine, 9248 (18.33%) guanosine, and 17,033 (33.76%) thymine. The genome contains 24 conserved core protein-coding genes, 25 tRNA genes, and 2 rRNA genes. Phylogenetic analysis based on the combined mitochondrial gene set showed that Rhizophagus sp. has a close relationship with Rhizophagus fasciculatus, Glomus irregular, and G. intratadices.

The complete mitochondrial genome of medicinal fungus Taiwanofungus camphoratus reveals gene rearrangements and intron dynamics of Polyporales.

Taiwanofungus camphoratus is a highly valued medicinal mushroom that is endemic to Taiwan, China. In the present study, the mitogenome of T. camphoratus was assembled and compared with other published Polyporales mitogenomes. The T. camphoratus mitogenome was composed of circular DNA molecules, with a total size of 114,922 bp. Genome collinearity analysis revealed large-scale gene rearrangements between the mitogenomes of Polyporales, and T. camphoratus contained a unique gene order. The number and classes of introns were highly variable in 12 Polyporales species we examined, which proved that numerous intron loss or gain events occurred in the evolution of Polyporales. The Ka/Ks values for most core protein coding genes in Polyporales species were less than 1, indicating that these genes were subject to purifying selection. However, the rps3 gene was found under positive or relaxed selection between some Polyporales species. Phylogenetic analysis based on the combined mitochondrial gene set obtained a well-supported topology, and T. camphoratus was identified as a sister species to Laetiporus sulphureus. This study served as the first report on the mitogenome in the Taiwanofungus genus, which will provide a basis for understanding the phylogeny and evolution of this important fungus.

Biosorption of sterols from tobacco waste extract using living and dead of newly isolated fungus Aspergillus fumigatus strain LSD-1.

Sterols are verified to be able to produce polycyclic aromatic hydrocarbons during its pyrolysis. In this study, a kind of Aspergillus fumigatus (LSD-1) was isolated from cigar leaves, and the biosorption effects on the stigmasterol, ß-sitosterol, campesterol, cholesterol, and ergosterol by using living and dead biomass of LSD-1 were investigated. The results showed that both living and dead biomass could efficiently remove these sterols in aqueous solution and tobacco waste extract (TWE). Interestingly, compared with the living biomass of LSD-1, the dead biomass of LSD-1 not only kept a high adsorption efficiency but also did not produce ergosterol. Overall, dead biomass of LSD-1 was a more suitable biosorbent to sterols in TWE. Furthermore, Brunner-Emmet-Teller (BET), Fourier transformed infrared spectrometer (FTIR) and scanning electron microscope (SEM) analysis were used to explore the biosorption process of living and dead biomass and their differences, suggesting that the biosorption of sterols was a physical process.

Antitumor Compounds from the Stout Camphor Mushroom Taiwanofungus camphoratus (Higher Basidiomycetes) Spent Culture Broth.

A known compound, 5-(hydroxymethyl) furan-2-carbaldehyde, and a novel compound, 3-isobutyl-1-methoxy-4-(4'-(3-methylbut-2-enyloxy)phenyl)-1H-pyrrole-2,5-dione were isolated from spent broth from submerged cultures of Taiwanofungus camphoratus. Their structures were elucidated by nuclear magnetic resonance (1H, 13C, and 2D) and mass spectra. These compounds inhibited the proliferation of K562 and HepG2 tumor cells in vitro.

Screening, Gene Cloning, and Characterizations of an Acid-Stable α-Amylase.

Based on its α-amylase activity at pH 5.0 and optimal pH of the crude enzyme, a strain (named B-5) with acid α-amylase production was screened. The B-5 strain was identified as Bacillus amyloliquefaciens through morphological, physiological, and biochemical characteristics analysis, as well as 16S rDNA phylogenetic analysis. Its α-amylase gene of GenBank Accession No. GU318401 was cloned and expressed in Escherichia coli. The purified recombinant α-amylase AMY-Ba showed the optimal pH of 5.0, and was stable at a pH range of 4.0-6.0. When hydrolyzing soluble starch, amylose, and amylopectin, AMY-Ba released glucose and maltose as major end products. The α-amylase AMY-Ba in this work was a different type from the well-investigated J01542 (GenBank Accession No.)-type α-amylase from the same species. AMY-Ba exhibited notable adsorption and hydrolysis ability towards various raw starches. Structure analysis of AMY-Ba suggested the presence of a new starch-binding domain at its C-terminal region.

Immobilization of Aspergillus niger xylanase on chitosan using dialdehyde starch as a coupling agent.

Dialdehyde starch (DAS) was used as a novel coupling agent to prepare chitosan carrier to immobilize the xylanase from Aspergillus niger A-25. Compared with glutaraldehyde-cross-linked chitosan (CS-GA) and pure chitosan beads, the DAS-cross-linked chitosan (CS-DAS) beads exhibited the highest xylanase activity recovery. The DAS adding amount and cross-linking time in CS-DAS preparation process were optimized with respect to activity recovery to the values of 1.0 g (6.7% w/v concentration) and 16 h, respectively. The optimum temperature of both the CS-DAS- and CS-GA-immobilized xylanase was observed to be 5 degrees C higher than that of free enzyme (50 degrees C). The CS-DAS-immobilized xylanase had the highest thermal and storage stability as compared to the CS-GA-immobilized and free xylanase. The apparent K (m) and V (max) values of the CS-DAS-immobilized xylanase were estimated to be 1.29 mg/ml and 300.7 mumol/min/mg protein, respectively. The CS-DAS-immobilized xylanase could produce from birchwood xylan high-quality xylo-oligosaccharides, mainly composed of xylotriose, as free xylanase did. The proposed CS-DAS carrier was more advantageous over the CS-GA or pure chitosan carrier for xylanase immobilization application.

[Overexpression of GST gene accelerates the growth of transgenic Arabidopsis under salt stress].

The Suaeda salsa glutathione s-transferase gene (GST) was inserted downstream of the 35S promoter in the plant expression vector pROK II and then was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens through floral dip method. Transformants were selected for their ability to grow on medium containing kanamycin. The fact that the GST gene had been transferred into the Arabidopsis thaliana genome was confirmed by the PCR-Southern blotting analysis. After cultivation, independent homozygous transgenic lines were obtained after selection of T(3) progenies on MS medium containing kanamycin. The expression of the gene transferred into the Arabidopsis thaliana was confirmed by Northern blotting. During salt stress, analysis of total glutathione (both oxidized and reduced type) and biomass of transgenic and wild Arabidopsis. The biomass of transgenic lines (GT) was slightly but significantly greater than that of wild type line (WT), and levels of oxidized glutathione (GSSG) were significantly higher in transgenic lines than in wild type. Therefore, overexpression of GST can increase Arabidopsis growth under salt stress, and this effect can be caused by oxidation of the reduced glutathione (GSH ).