A continuous thermal lysis procedure for the large-scale preparation of plasmid DNA

There is an increasing interest and need for the development of scaleable process for the preparation of plasmid DNA for vaccines and gene therapy. In this report, we describe a streamline modified process of plasmid extraction based on boiling lysis in order to simplify the operation and process large volumes of Escherichia coli cultures. The bacteria, harvested using a hollow fiber cartridge after fermentation, were treated with lysozyme at 37 degrees C prior to passing through a heat-exchanger coil. Subsequently, the supernatant was separated from lysed bacteria using a 65 microm nylon filter. The employment of a peristaltic pump and two heating coils at constant temperature without the use of centrifugation enabled the process protocol to be constant and controllable. A relatively low lysis temperature of approximately 70-80 degrees C and a buffer modified for the high-density cultures were also optimized for the process. Prior to thermal lysis, a pre-treatment step with the lysozyme for 20 min at 37 degrees C was one of the crucial steps contributing to the high plasmid quantity and quality from batch to batch. After harvesting 17 L of E. coli cultures (OD600 = 50), the plasmid can be extracted within 45 min with this streamline protocol. The plasmid yields are approximately 100mg/L culture, which makes it attractive and promising for the large-scale preparation of plasmid.     

Semiautomated preparation of DNA templates for large-scale sequencing projects

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

A computational study of nucleosomal DNA flexibility

Molecular dynamics simulations of the nucleosome core particle and its isolated DNA free in solution are reported. The simulations are based on the implicit solvent methodology and provide insights into the nature of large-scale structural fluctuations and flexibility of the nucleosomal DNA. In addition to the kinked regions previously identified in the x-ray structure of the nucleosome, the simulations support the existence of a biochemically identified distorted region of the DNA. Comparison of computed relative free energies shows that formation of the kinks is associated with little, if any, energy cost relative to a smooth, ideal conformation of the DNA superhelix. Isolated nucleosomal DNA is found to be considerably more flexible than expected for a 147 bp stretch of DNA based on its canonical persistence length of 500 A. Notably, the significant bending of the DNA observed in our simulations occurs without breaking of Watson-Crick bonds. The computed relative stability of bent conformations is sensitive to the ionic strength of the solution in the physiological range; the sensitivity suggests possible experiments that might provide further insights into the structural origins of the unusual flexibility of the DNA.     

Flexibility of nucleosomal DNA

In this study transient electric birefringence (TEB) has been used to investigate the molecular flexibility of short fragments of DNA. Nucleosomal DNA always exhibits negative birefringence and Kerr behavior was observed up to high field strengths (6 KV/cm). The value of the Kerr constant is 3.5 10^?2 e.s.u.. Birefringence decays were single exponentials and a field dependence of the molecular orientational relaxation time τ was found: it is explained by an inherent flexibility of the DNA molecule. A 20 % decrease in the calculated length was observed with fields applied as low as 2 KV/cm. The results obtained at very low fields establish TEB as a method well suited to calculate accurate values for the length of small fragments of DNA: the τ value of 4.3 μsec corresponds to a DNA length of 660 Å.     

A new vector for insertion of any DNA fragment into the chromosome of transformable Neisseriae

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.     

Rapid spontaneous accessibility of nucleosomal DNA

DNA wrapped in nucleosomes is sterically occluded, creating obstacles for proteins that must bind it. How proteins gain access to DNA buried inside nucleosomes is not known. Here we report measurements of the rates of spontaneous nucleosome conformational changes in which a stretch of DNA transiently unwraps off the histone surface, starting from one end of the nucleosome, and then rewraps. The rates are rapid. Nucleosomal DNA remains fully wrapped for only approximately 250 ms before spontaneously unwrapping; unwrapped DNA rewraps within approximately 10-50 ms. Spontaneous unwrapping of nucleosomal DNA allows any protein rapid access even to buried stretches of the DNA. Our results explain how remodeling factors can be recruited to particular nucleosomes on a biologically relevant timescale, and they imply that the major impediment to entry of RNA polymerase into a nucleosome is rewrapping of nucleosomal DNA, not unwrapping.     

Structure and dynamics of nucleosomal DNA

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.     

Geometry of the nucleosomal DNA superhelix

Nucleosome stability is largely an indirect measure of DNA sequence based on the material properties of DNA and the ability of a sequence to assume the required left-handed superhelical conformation. Here we focus attention only on the geometry of the superhelix and present two distinct mathematical expressions that rely on the DNA helical parameters (Shift, Slide, Rise, Tilt, Roll, Twist). One representation requires torsion for superhelix formation; the other requires shear. To compare these mathematical expressions to experimental data we develop a strategy for Fourier-filtering the helical parameters that identifies necessary and sufficient conditions to achieve a high-resolution model of the nucleosome superhelix. We apply this filtering strategy to 24 high-resolution structures of the nucleosome and demonstrate that all structures have a highly conserved distribution of Roll, Slide and Twist that involves two length scales. One length scale spans the entire length of nucleosomal DNA. The other is associated with the helix repeat. Our strategy also enables us to identify ground state or simple nucleosomes and altered nucleosome structures. These results form a basis for characterizing structural variations in the emerging family of nucleosome structures and a method for further developing structure-based models of nucleosome stability.     

Heat-labile phosphatase simplifies the preparation of dephosphorylated vector DNA

A novel enzyme for DNA dephosphorylation, HK phosphatase, is completely and irreversibly inactivated at 65 degrees C. HK phosphatase treatment of BamHI-digested pBR322 reduces nonrecombinants in cloning experiments to approx. 5% of transformant colonies.     

A simple method for large-scale preparation of chick embryo tRNA

A simple method for preparing bulk quantities of tRNA from chick embryo has been developed. In this method chick embryos were homogenized in a buffer of pH 4.5, followed by deproteinization with phenol. The aqueous layer was allowed to separate under gravity. The resulting aqueous layer, after two more phenol treatments, was directly passed through a DEAE-cellulose column and the tRNA eluted therefrom with 1 m NaCl. The tRNA prepared by this method was as active as the one prepared at neutral pH.     

Working the kinks out of nucleosomal DNA

Condensation of DNA in the nucleosome takes advantage of its double-helical architecture. The DNA deforms at sites where the base pairs face the histone octamer. The largest so-called kink-and-slide deformations occur in the vicinity of arginines that penetrate the minor groove. Nucleosome structures formed from the 601 positioning sequence differ subtly from those incorporating an AT-rich human α-satellite DNA. Restraints imposed by the histone arginines on the displacement of base pairs can modulate the sequence-dependent deformability of DNA and potentially contribute to the unique features of the different nucleosomes. Steric barriers mimicking constraints found in the nucleosome induce the simulated large-scale rearrangement of canonical B DNA to kink-and-slide states. The pathway to these states shows nonharmonic behavior consistent with bending profiles inferred from AFM measurements.     

Predicting preferential DNA vector insertion sites: implications for functional genomics and gene therapy

Viral and transposon vectors have been employed in gene therapy as well as functional genomics studies. However, the goals of gene therapy and functional genomics are entirely different; gene therapists hope to avoid altering endogenous gene expression (especially the activation of oncogenes), whereas geneticists do want to alter expression of chromosomal genes. The odds of either outcome depend on a vector's preference to integrate into genes or control regions, and these preferences vary between vectors. Here we discuss the relative strengths of DNA vectors over viral vectors, and review methods to overcome barriers to delivery inherent to DNA vectors. We also review the tendencies of several classes of retroviral and transposon vectors to target DNA sequences, genes, and genetic elements with respect to the balance between insertion preferences and oncogenic selection. Theoretically, knowing the variables that affect integration for various vectors will allow researchers to choose the vector with the most utility for their specific purposes. The three principle benefits from elucidating factors that affect preferences in integration are as follows: in gene therapy, it allows assessment of the overall risks for activating an oncogene or inactivating a tumor suppressor gene that could lead to severe adverse effects years after treatment; in genomic studies, it allows one to discern random from selected integration events; and in gene therapy as well as functional genomics, it facilitates design of vectors that are better targeted to specific sequences, which would be a significant advance in the art of transgenesis.     

A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.