Extraction of total RNA from adipocytes.

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 degrees C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 microg total RNA from 2 x 10(6) cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.     

Purification of bacteriophage M13 by anion exchange chromatography

M13 is a non-lytic filamentous bacteriophage (phage). It has been used widely in phage display technology for displaying foreign peptides, and also for studying macromolecule structures and interactions. Traditionally, this phage has been purified by cesium chloride (CsCl) density gradient ultracentrifugation which is highly laborious and time consuming. In the present study, a simple, rapid and efficient method for the purification of M13 based on anion exchange chromatography was established. A pre-packed SepFast™ Super Q column connected to a fast protein liquid chromatography (FPLC) system was employed to capture released phages in clarified Escherichia coli fermented broth. An average yield of 74% was obtained from a packed bed mode elution using citrate buffer (pH 4), containing 1.5 M NaCl at 1 ml/min flow rate. The purification process was shortened substantially to less than 2 h from 18 h in the conventional ultracentrifugation method. SDS-PAGE revealed that the purity of particles was comparable to that of CsCl gradient density ultracentrifugation method. Plaque forming assay showed that the purified phages were still infectious.     

Purification and characterization of the gag gene products of avian-type C retroviruses by high-pressure liquid chromatography

We have developed an inexpensive procedure for bacteriophage purification suitable for small- and medium-scale preparations (up to one liter of lysate). The method consists of precipitation with polyethylene glycol 6000 and gel chromatography on a Bio-Gel A-5m column. The purity of the phage preparation is comparable to that obtained by CsCl step gradient ultracentrifugation.     

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing practices for recombinant adeno‐associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10⁴–10⁵ vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small‐scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol‐step gradient whereas large‐scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead‐end filtration) and chromatography based‐methods are used for large‐scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.     

Comparison of different myelin isolation methods by use of nonequilibriumpH gradient gel electrophoresis

Myelin was isolated from bovine white matter by five published procedures and several modifications of two of them. Comparison of the protein profiles of the preparations by nonequilibrium pH gradient gel electrophoresis, revealed clear differences in myelin protein content and composition between preparations obtained by different methods. In isolation methods where the medium contained salts, some of the myelin proteins were solubilized, the phenomenon being most pronounced in long-period isolations in buffered CsCl solution.     

Efficient and rapid purification of drug- and gene-carrying bio-nanocapsules, hepatitis B virus surface antigen L particles, from Saccharomyces cerevisiae

Bio-nanocapsules (BNCs) are hollow particles (approx. 50 nm diameter) consisting of hepatitis B virus surface antigen (HBsAg) large (L, pre-S1+pre-S2+S) proteins embedded in a unilamellar liposome, sharing the same transmembrane S region with an immunogen of hepatitis B vaccine (i.e., HBsAg small (S) protein particle). BNCs can incorporate drugs and genes into the hollow space and systemic administration of the BNCs can deliver the products to human liver via the human hepatocyte-specific receptor within the pre-S (pre-S1+pre-S2) region displayed on BNC's surface. Thus, BNCs are expected to offer efficient and safe non-viral nanocarriers to deliver human liver-specific genes and drugs. To date, BNCs have been purified from the crude extract of BNC-overexpressing yeast cells by fractionation with polyethylene glycol followed by one CsCl equilibrium and two sucrose density gradient ultracentrifugation steps. However, the process was inefficient in terms of yield and time, and was not suitable for mass production because of the ultracentrifugation step. Furthermore, trace contamination with yeast-derived proteinases degraded the pre-S region, which is indispensable for liver-targeting, during long-term storage. In this study, we developed a new purification method involving heat treatment and sulfated cellulofine column chromatography to facilitate rapid purification, completely remove proteinases, and enable mass production. In addition, the BNCs were functional for at least 14 months after lyophilization with 5% (w/v) sucrose as an excipient. This new process will significantly contribute to the development of forthcoming BNC-based nanomedicines as well as hepatitis B vaccines.     

Physical properties of a genomic condensed chromatin fragment

We have studied the physical properties of a segment of condensed chromatin that lies upstream of the chicken beta-globin locus. This segment can be excised from an avian erythroleukemia cell line by restriction enzyme digestion and released from the nucleus as an essentially homogeneous fragment about 15.5 kbp long. Because of this homogeneity we could measure its sedimentation coefficient quite accurately by a combination of sucrose gradient and analytical ultracentrifugation. By measuring additionally the buoyant density of the cross-linked particle in CsCl we could deduce the total mass of the particle, hence its frictional coefficient, f, directly related to its shape. The measured value of f is consistent with a rod-like particle of the approximate length and diameter proposed earlier for the 30 nm chromatin fiber. The method is generally applicable to homogeneous particles of unique sequence at genomic abundance.     

FRACTIONATION OF NUCLEAR,CHLOROPLAST, AND MITOCHONDRIAL DNA FROM POLYSIPHONIA BOLDII (RHODOPHYTA) USING A RAPID AND SIMPLE METHOD FOR THE SIMULTANEOUS ISOLATION OF RNA AND DNA1

Extraction of nucleic acids from red algae is complicated by the presence of phycocolloids. For this reason, methods used for nucleic acid isolation from other organisms are not always amenable to use with red algal preparations; modifications in some cases lead to protocols that are time consuming and complicated, often requiring large amounts of algal tissue for starting material. Here we describe the isolation of both RNA and DNA followed by fractionation and identification of nuclear, chloroplast, and mitochondrial DNAs from a single preparation of Polysiphonia boldii Wynne and Edwards using a simple method that yielded approximately 100 μg of total RNA and 20 μg of total DNA from 1 g of frozen powdered algae. The potent protein denaturant guanidinium thiocyanate and the detergent sarkosyl were used to gently lyse the cells and organelles and immediately inhibit nuclease activity in the extract. The nucleic acids were isolated by ultracentrifugation into a dense solution of CsCl; the RNA was recovered as a pellet and the DNA as a band within the CsCl solution. Agarose gel electrophoresis of the total RNA showed discrete ribosomal RNA bands, indicating little nonspecific degradation. The nuclear, chloroplast, and mitochondrial DNAs were fractionated by density gradient ultracentrifugation in the presence of the DNA binding dye, bisbenzimide H (Hoechst 33258), which binds preferentially to DNA with a high A + T:G + C ratio, thus altering its density to a greater degree than it does that of DNA with a lower nucleotide ratio. The three fractions were identified by Southern blot analysis using heterologous gene probes specific for the different genomes. The protocol should be applicable to different types of algae. The simple nucleic acid isolation step can be performed on multiple samples simultaneously without subsequent fractionation of DNA, allowing comparison of DNA from different individuals, populations, or species.     

Optimizing Phytoplasma DNA purification for genome analysis.

Genome analysis of uncultivable plant pathogenic phytoplasmas is hindered by the difficulty in obtaining sufficient quantities of phytoplasma enriched DNA. We investigated a combination of conventional enrichment techniques such as cesium chloride (CsCl) buoyant gradient centrifugation, and new methods such as rolling circle amplification (RCA), suppression subtractive hybridization (SSH), and mirror orientation selection (MOS) to obtain DNA with a high phytoplasma:host ratio as the major first step in genome analysis of Candidatus Phytoplasma australiense. The phytoplasma:host ratio was calculated for five different plasmid libraries. Based on sequence data, 90% of clones from CsCl DNA enrichment contained chromosomal phytoplasma DNA, compared to 60% from RCA CsCl DNA and 20% from SSH subtracted libraries. Based on an analysis of representative libraries, none contained plant DNA. A high percentage of clones (80-100%) from SSH libraries contained extrachromosomal DNA (eDNA), and we speculate that eDNA in the original DNA preparation was amplified in subsequent SSH manipulations. Despite the availability of new techniques for nucleic acid amplification, we found that conventional CsCl gradient centrifugation was the best enrichment method for obtaining chromosomal phytoplasma DNA with low host DNA content.     

Rapid step-gradient purification of mitochondrial DNA

A convenient modification of the step gradient (CsCl/ethidium bomide) procedure is described. This rapid method allows isolation of covalently closed circular DNA separated from contaminating proteins, RNA and chromosomal DNA in ca. 5 h. Large scale preparations can be performed for circular DNA from eukaryotic organelles (mitochondria). The protocol uses organelle pelleting/NaCl-sarcosyl incubation steps for mitochondria followed by a CsCl step gradient and exhibits yields equal to the conventional procedures. It results in DNA sufficiently pure to be used for restriction endonuclease analysis, subcloning, 5-end labeling, gel retention assays, and various types of hybridization.     

Isolation of DNA-free RNA, DNA, and proteins by cesium trifluoroacetate centrifugation

The ability to simultaneously isolate intact DNA-free RNA, genomic DNA, and proteins from a biological specimen can be useful in cloning genes and analyzing gene expression. Equilibrium density gradient centrifugation with CsCl is a useful tool for fractionating, quantitatively separating, and characterizing RNA, DNA, and the total quota of proteins, respectively, based on differences in their buoyant densities. In the present study we have reexamined the rarely used cesium salt, cesium trifluoroacetate, for the same purpose. A significant advantage of CsTFA lies in the fact that, unlike in CsCl, RNA can be recovered from a single, soluble fraction of the CsTFA gradient. Furthermore, unlike CsCl, CsTFA is freely soluble in ethanol so that co-precipitation of the salt in the recovered RNA upon alcohol precipitation does not take place. Hence, the RNA is recovered with minimum manipulations. The one-step separation of cellular macromolecule classes free of each other in small amount of starting materials provides a major advantage over other methods currently in use.     

小麦印度腥黑粉菌线粒体DNA提取及其ATP6基因在真菌遗传发育分析中的应用

小麦印度腥黑粉菌(Tilletia indica Mitra)是一种世界范围的重要检疫性有害真菌,该病原菌和近似种之间冬孢子的形态特征极为相似,遗传关系非常相近。为了从分子水平上探讨小麦印度腥黑粉菌和近似种之间线粒体基因序列的差异,从新鲜菌丝中提取总DNA,经两次氯化铯密度梯度超速离心分离线粒体DNA(mtDNA),提取的mtDNA纯度较高,可用于克隆、酶切分析和PCR扩增等分析。选取基因ATP(adenosine triphosphate)6的序列,并结合GenBank中相关种类的ATP6基因DNA序列进行了系统发育分析。结果表明,线粒体基因ATP6可用于科属水平的分类鉴定。     

The influence of environmental agents on prostaglandin biosynthesis and metabolism in the lung. Inhibition of lung 15-hydroxyprostaglandin dehydrogenase by exposure of guinea pigs to 100 per cent oxygen at atmospheric pressure.

The methods of Hascall & Sajdera (1969) were used to compare the proteoglycans of human intervertebral disc with those of bovine nasal cartilage. In contrast with cartilage, most of the hexuronate of disc could be extracted at low shear with water or dilute salt solutions. Extracts of disc with 4M-guanidinium chloride were centrifugated in 0.4M-guanidinium chloride in a CsCl gradient. Analytical ultracentrifugation of the hexuronate-containing heavy component revealed two fractions. both more polydisperse than those of cartilage. Also the more rapidly sediminting component was a much smaller fraction of the total. After prior extraction with 0.4M-guanidinium chloride, 4M-guanidinium chloride extracts of disc were found, by ultracentrifugal analysis, to be enriched in components resembling the proteoglycan monomer and aggregating factors of cartilage.     

Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis

Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein, we present here a tethered catalysis approach for the production of acetylated p53 in vivo. P53 is a major tumor suppressor protein that protects the cell from various oncogenic stresses. Upon DNA damage, p53 is stabilized and activated by a plethora of post-translational modifications, including acetylation. Here, we show that constitutively acetylated p53 can be expressed and purified from both yeast and Escherichia coli. This method is highly suitable for studying protein-protein interactions in the conventional yeast two-hybrid screen that requires a constitutively acetylated state of p53. Furthermore, effective production and purification of acetylated p53 from E. coli supports future biochemical and structural characterization. The method described in this work can be applied to other proteins and modifications, and thus has widespread use in the fields of signal transduction and proteomic research.     

Intracellular forms of simian virus 40 nucleoprotein complexes. II. Biochemical and electron microscopic analysis of simian virus 40 virion assembly.

The simian virus 40 virion assembly process was studied with pulse-labeling kinetics of virion proteins, CsCl gradient analysis, electron microscopy, and low-salt gel electrophoresis. The results obtained are consistent with the model of gradual addition and organization of capsid proteins around simian virus 40 chromatin. Empty virions, as observed in the CsCl gradient by previous workers, were found to be the dissociation product of immature virus. Histone H1 was found in simian virus 40 chromatin and virion assembly intermediates but not in the mature virion banding at 1.34 g/ml in the CsCl gradient.     

Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.     

Lymphocyte-activating factor. I. Generation and physicochemical characterization.

Lipopolysaccharide-activated murine peritoneal macrophages elaborate lymphocyte-activating factor (LAF) which is mitogenic for murine thymocytes. A method of LAF production is presented that permits the generation of a relatively homogeneous molecular species. LAF has an isoelectric point of 4.8 (range 4.7-4.9). The m.w. was determined by using several physical techniques. The apparent sedimentation coefficient (S20,w) was determined to be 2.0S by sucrose gradient ultracentrifugation. The Stokes (molecular) radius was determined by gel filtration on Sephadex G-75 to be 22 A (range 21.5 to 22.5); the calculated diffusion coefficient (D20,w) was 9.7 X 10(-7) cm2/sec (range 9.5 X 10(-7) to 9.9 X 10(-7). The buoyant density of LAF is 1.30 g/cm3 (range 1.27 to 1.33) as determined by CsCl isopycnic ultracentrifugation; the partial specific volume was estimated to be 0.72 (range 0.70 to 0.74). From these data, the m.w. was calculated to be 18,000 daltons (range 16,400 to 19,600) with the Svedberg equation. The frictional ratio was calculated to be 1.25.     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.