Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

Transfer and Expression of a Multiple Antibiotic Resistance Plasmid in Marine Bacteria

Conjugal transfer of a multiresistance plasmid from Pseudomonas fluorescens to halophilic and halotolerant bacteria was studied under in vitro and in situ conditions. Mating conducted in broth as well as on plates yielded a plasmid transfer frequency of as high as 10⁻³. Among these two, plate mating facilitated conjugal transfer of plasmid, because the cell-to-cell contact is more in plate mating. When P. fluorescens was incubated in seawater, the organism progressively lost its colony forming activity within 15 days. Microscopic examination revealed the presence of very short rods, indicating that the cells have become viable but nonculturable (VNC). Mating conducted in natural seawater without any added nutrients revealed that the conjugal transfer is influenced by the physical state of the donor and the recipients as well as the availability of nutrients. But a plasmid transfer frequency of 10⁻⁷ was obtained even after the donor cells have become VNC suggesting that the nonculturable state and nutrient deprived condition may not limit plasmid transfer. The results suggest that the terrestrial bacteria entering into the seawaters with antibiotic resistance plasmids may be responsible for the prevalence of resistance genes in the marine environment. Received: 4 May 1998 / Accepted: 18 June 1998     

Comparative study on enzymatic digestibility of switchgrass varieties and harvests processed by leading pretreatment technologies

Feedstock quality of switchgrass for biofuel production depends on many factors such as morphological types, geographic origins, maturity, environmental and cultivation parameters, and storage. We report variability in compositions and enzymatic digestion efficiencies for three cultivars of switchgrass (Alamo, Dacotah and Shawnee), grown and harvested at different locations and seasons. Saccharification yields of switchgrass processed by different pretreatment technologies (AFEX, dilute sulfuric acid, liquid hot water, lime, and soaking in aqueous ammonia) are compared in regards to switchgrass genotypes and harvest seasons. Despite its higher cellulose content per dry mass, Dacotah switchgrass harvested after wintering consistently gave a lower saccharification yield than the other two varieties harvested in the fall. The recalcitrance of upland cultivars and over-wintered switchgrass may require more severe pretreatment conditions. We discuss the key features of different pretreatment technologies and differences in switchgrass cultivars and harvest seasons on hydrolysis performance for the applied pretreatment methods.     

Enzyme characterization for hydrolysis of AFEX and liquid hot-water pretreated distillers' grains and their conversion to ethanol

Dried distillers' grains with solubles (DDGS), a co-product of corn ethanol production, was investigated as a feedstock for additional ethanol production. DDGS was pretreated with liquid hot-water (LHW) and ammonia fiber explosion (AFEX) processes. Cellulose was readily converted to glucose from both LHW and AFEX treated DDGS using a mixture of commercial cellulase and beta-glucosidase; however, these enzymes were ineffective at saccharifying the xylan present in the pretreated DDGS. Several commercial enzyme preparations were evaluated in combination with cellulase to saccharify pretreated DDGS xylan and it was found that adding commercial grade (e.g. impure) pectinase and feruloyl esterase (FAE) preparations were effective at releasing arabinose and xylose. The response of sugar yields for pretreated AFEX and LHW DDGS (6wt%/solids) were determined for different enzyme loadings of FAE and pectinase and modeled as a response surfaces. Arabinose and xylose yields rose with increasing FAE and pectinase enzyme dosages for both pretreated materials. When hydrolyzed at 20wt%/solids with the same blend of commercial enzymes, the yields were 278 and 261g sugars (i.e. total of arabinose, xylose, and glucose) per kg of DDGS (dry basis, db) for AFEX and LHW pretreated DDGS, respectively. The pretreated DDGS's were also evaluated for fermentation using Saccharomyces cerevisiae at 15wt%/solids. Pretreated DDGS were readily fermented and were converted to ethanol at 89-90% efficiency based upon total glucans; S. cerevisiae does not ferment arabinose or xylose.     

Gene Expression Analysis in Sorghum Hybrids and Their Parental Lines at Critical Developmental Stages in Relation to Grain Yield Heterosis by Exploiting Heterosis-Related Genes from Major Cereals

Relative expression levels of selected genes from the Heterosis-Related Gene Database exhibiting more than 90% homology with sorghum were studied in hybrids and their respective parental lines for a better understanding on the molecular basis of heterosis. A high (27A × RS 673) and a low heterotic hybrid (7A × CB 26) of sorghum along with their parental lines were used for this purpose. Twenty (15 maize and 5 rice) genes exhibiting more than 90% homology with that of sorghum were identified. The maize genes ZmHG13, ZmHG16, and ZmhG19 exhibited more than fourfold increase over the male parent (RS 673) of high heterotic hybrid during booting stage, which started decreasing during flowering stage. Similarly, the rice genes OsHG1 and OsHG12 recorded >?2.5-fold increase. However, these genes recorded less than twofold increase during the same stage of the plant in the low heterotic hybrid. Notably, among the genes that exhibited higher expression in the highly heterotic hybrid were those coding for proteins, which were known to play crucial roles in the manifestation of heterosis in plants.     

Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants

Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.     

Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ～1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ～85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95–98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX‐treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX‐treated bagasse. Co‐fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX‐treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH‐ST) produced 34–36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. Biotechnol. Bioeng. 2010;107: 441–450. © 2010 Wiley Periodicals, Inc.     

A cross-cancer metastasis signature in the microRNA–mRNA axis of paired tissue samples

In the progression of cancer, cells acquire genetic mutations that cause uncontrolled growth. Over time, the primary tumour may undergo additional mutations that allow for the cancerous cells to spread throughout the body as metastases. Since metastatic development typically results in markedly worse patient outcomes, research into the identity and function of metastasis-associated biomarkers could eventually translate into clinical diagnostics or novel therapeutics. Although the general processes underpinning metastatic progression are understood, no clear cross-cancer biomarker profile has emerged. However, the literature suggests that some microRNAs (miRNAs) may play an important role in the metastatic progression of several cancer types. Using a subset of The Cancer Genome Atlas (TCGA) data, we performed an integrated analysis of mRNA and miRNA expression with paired metastatic and primary tumour samples to interrogate how the miRNA–mRNA regulatory axis influences metastatic progression. From this, we successfully built mRNA- and miRNA-specific classifiers that can discriminate pairs of metastatic and primary samples across 11 cancer types. In addition, we identified a number of miRNAs whose metastasis-associated dysregulation could predict mRNA metastasis-associated dysregulation. Among the most predictive miRNAs, we found several previously implicated in cancer progression, including miR-301b, miR-1296, and miR-423. Taken together, our results suggest that metastatic samples have a common cross-cancer signature when compared with their primary tumour pair, and that these miRNA biomarkers can be used to predict metastatic status as well as mRNA expression.

     

Antigen is required for maturation and activation of pathogenic anti-DNA antibodies and systemic inflammation

Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies and systemic inflammation that results in part from dendritic cell activation by nucleic acid containing immune complexes. There are many mouse models of lupus, some spontaneous and some induced. We have been interested in an induced model in which estrogen is the trigger for development of a lupus-like serology. The R4A transgenic mouse expresses a transgene-encoded H chain of an anti-DNA Ab. This mouse maintains normal B cell tolerance with deletion of high-affinity DNA-reactive B cells and maturation to immunocompetence of B cells making nonglomerulotropic, low-affinity DNA-reactive Abs. When this mouse is given estradiol, normal tolerance mechanisms are altered; high-affinity DNA-reactive B cells mature to a marginal zone phenotype, and the mice are induced to make high titers of anti-DNA Abs. We now show that estradiol administration also leads to systemic inflammation with increased B cell-activating factor and IFN levels and induction of an IFN signature. DNA must be accessible to B cells for both the production of high-affinity anti-DNA Abs and the generation of the proinflammatory milieu. When DNase is delivered to the mice at the same time as estradiol, there is no evidence for an abrogation of tolerance, no increased B cell-activating factor and IFN, and no IFN signature. Thus, the presence of autoantigen is required for positive selection of autoreactive B cells and for the subsequent positive feedback loop that occurs secondary to dendritic cell activation by DNA-containing immune complexes.