Article

Rapid Purification of Plasmid DNAs by Hydroxyapatite Chromatography

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Abstract

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 × 106. This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.

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... as described by Clewell (1972) with modifications according to Teather et al. (1978). Plasmid DNA on a preparative scale was purified by chromatography on hydroxyapatite (Colman et al., 1978) and, when the DNA had to be pure (in our hands the latter method gave DNA in excellent yield but never truly free from chromosomal DNA), by subsequent CsC1 density gradient centrifugation. Restriction endonucleases were from Boehringer (BamHI, EeoRI, SmaI, HindIII) or from New England Biolabs (PvuII, XhoI), DNA ligase was from Miles. ...
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pTU 100 is a hybrid plasmid constructed by cloning a 7.5 Kb EcoRI fragment (carrying the wildtype ompA gene) onto pSC 101 (Henning et al., 1979). This plasmid confers sensitivity to phages Tull* and K3h1 when present in an ompA host strain, due to the expression of the phage receptor protein II* from the plasmid ompA + gene. Plasmid mutants have been isolated that have become resistant to one or both of these phages. Restriction endonuclease analysis and DNA-sequencing studies in these plasmids demonstrate that a BamHI site and two PvuII sites are located within the ompA gene. BamHI cuts the gene at a site corresponding to residue 227 within a total of 325 amino acid residues.Neither the wildtype ompA gene nor the BamHI fragment encoding the NH2-terminal part of the protein (residues 1–227) could be transferred to a high copy number plasmid, presumably due to lethal overproduction of the protein or its NH2-terminal fragment. However, the NH2-terminal fragment derived from one of the ompA mutants of pTU100 could be transferred to the high copy number plasmid pBR322, and was expressed in the presence of the amber suppressors supD or supF. Under these conditions two new envelope proteins with apparent molecular weights of 30,000 and 24,000 were synthesized, and the cells became sensitive to phage TuII*, indicating the presence of phage receptor activity in the outer membrane. The major, 24,000 dalton protein has the molecular weight expected of a protein comprising residues 1–227 of protein II*. DNA-sequencing studies demonstrated that no termination codons are present in the DNA region immediately downstream from the BamHI site at residue 227 in this hybrid plasmid, and it is therefore likely that the 24,000-dalton protein arises from the posttranslational proteolytic cleavage of a larger polypeptide. The 30,000-dalton protein is a likely candidate for such a larger polypeptide. These results also demonstrate that the 98 CO2H-terminal residues of wildtype protein II* (resisdues 228–325) are not required either for the activity of the protein as a phage receptor or for its incorporation into the outer membrane.
... The method that use silica oxide as the DNAbinding matrix has the limitation, as bacterial lipopolysaccharide or endotoxin is co-purified which can interfere with downstream applications. The lipopolysaccharide content in plasmid preparation can heavily influence the efficiency of transfection (Clevell, and Helinski, 1969, Colman et.al 1978, Barnes, 1977). ...
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Isolation of quality plasmid DNA from bacterial cells is critical in molecular biology experiments. Although efficient protocol for plasmid isolation are developed and standardized, rapid and cost effective protocols are desirable while handling a large number of samples. In present study we are reporting a simple procedure for extracting naked plasmid DNA which can be used for a large number of samples with sufficient purity. The method involves denaturation of high molecular weight chromosomal DNA using strong alkali, during neutralization process, chromosomal DNA renatures, and proteins denature to form an insoluble clot in the presence of choatropic agent guanidine hydrochloride, leaving plasmid DNA in the supernatant. The modified protocol which is less expensive compared to the commercial plasmid DNA isolation kit available, yields quality plasmid DNA. We tested the yield and quality of different kinds of plasmid extracted from bacteria. The results suggest that the protocol can be effectively used for molecular cloning, sequencing, and transfection.
... Plasmid extraction from cultured Escherichia coli strains is probably one of the most common laboratory practices. In the late 1960s, the first protocols for isolation of plasmid DNA were published [19][20][21], from which the alkaline lysis of bacterial cells in a slightly modified form became today's primarily used method [17,22,23]. Numerous commercial kits are based on this technology, which employs either silica-packed columns or silica-coated magnetic beads. ...
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Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
... Plasmid DNA isolation for cloning and protein expression has been in use for decades [1][2][3] and remains one of the most common methods used by molecular biologists. Plasmid purification involves two steps: bacterial lysis and DNA isolation. ...
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Plasmid purification is a basic tool of molecular biologists. Although the development of plasmid isolation kits utilizing silica spin columns reduced the time and labor spent on plasmid purification, achieving large plasmid DNA yields still requires significant time and effort. Here we introduce the Miraprep, a rapid protocol that allows isolation of plasmid DNA using commercial Miniprep kits, but with DNA yields comparable to commercial Maxiprep plasmid purifications. Combining ethanol precipitation with spin column purification, we created a DNA isolation protocol that yields highly concentrated plasmid DNA samples in less than 30 minutes. We show that Miraprep isolated plasmids are as stable as plasmids isolated by standard procedures, can be used for standard molecular biology procedures including DNA sequencing, and can be efficiently transfected into mammalian cells. This new plasmid DNA isolation protocol will significantly reduce time and labor without increasing costs.
... The crude lysates were prepared according to the method of Guerry et al. (1973). Plasmid DNA was purified on a hydroxylapatite column (Colman et al., 1978) followed by phenol treatment and Sephadex G50 column chromatography. Recombinant plasmids were screened by the alkali extraction procedure of Birnboim and Doly (1979). ...
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Direct expression of hepatitis B virus (HBV) surface antigen (HBsAg) gene under the control of the Escherichia coli tryptophan operon (trp) promoter has been achieved. Synthesis of HBsAg (both complete and lacking its N-terminal segment) as a part of hybrid proteins with the N-terminal portion coded by genes cat, kan or bla is controlled by the appropriate promoters, as well as by the trp promoter. The highest levels of expression, including those for direct synthesis of HBsAg, provide the accumulation of about 10(5) polypeptide molecules per bacterial cell.
... Plasmid DNA was isolated either by the hydroxylapatite method of Colman et AL. (10), or by CsCl-ethidium bromide density gradient centrifugation (8). ...
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... The pure pDNA could be produced by a multistep procedure-alkaline lysis, clarification/concentration, and purification process-following the collection of cells from fermentation media[1]. There are several choices for down-stream processing, especially the highly preferred chromatographic techniques such as anion-exchange chromatography (AEC)[2][3][4][5], size-exclusion chromatography (SEC)[6,7], hydroxyapatite chromatography (HAC)[8], affinity chromatography (AC)[9], thiophilic affinity chromatography (TAC)[10], hydrophobic interaction chromatography (HIC)[11][12][13][14][15]and nonchromatographic methods. These methods can be applied alone or in combination for this purpose. ...
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Chapter
By now, almost anyone involved in the biological sciences has gone through the process of purifying plasmid DNA from a bacterial host organism to use as a tool in molecular biology. The methodology has become so routine that it is common to purchase one of any number of kits that are designed to make the procedure as simple and as painless as possible. The evolution of plasmid preparation has come to the point that one can achieve success on the first try without having a solid foundation in the process fundamentals.
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It is generally accepted that hydroxyl radicals produced by the action of ionizing radiation in dilute aqueous solutions are predominantly responsible for the induction of strand breaks in DNA. It is also known that Tris buffer and citrate buffer can act as OH-scavengers. Nevertheless, Tris is still used as a solvent for DNA in irradiation experiments, even very recently, as can be seen from the work of McCormack et al. and Belli, reported during this meeting. We therefore considered it appropriate to study the OH-scavenging capacity of different buffers by measuring the DNA strand break production after ionizing radiation, starting with Tris buffer and phosphate buffer. Furthermore, we wanted to know whether drugs, known to interact with the DNA molecule, affect the radiation induced degradation and whether there is any correlation to the binding properties. Such knowledge might be important for the formulation of safety standards.
Chapter
A collection of 2500 clones containing hybrid plasmids representative of nearly the entire genome of B. subtilis 168 was established in E. coli SK1592 by using the poly(dA).poly(dT) joining method with randomly sheared DNA fragments and plasmid pHV33, a bifunctional vector which can replicate in both E. coli and B. subtilis. Thirty clones of the collection were shown to hybridize specifically with a B. subtilis rRNA probe. Recombinant plasmids extracted from E. coli were used to transform auxotrophic mutants of B. subtilis. Complementation was observed for several markers such as thr, leuA, hisA, glyB and purB. In B. subtilis rec+ strains in most cases, markers carried by the recombinant plasmids were lost from the plasmid and integrated into the chromosomal DNA. Such loss did not occur when a rec− strain harboring the marker recE4 was used.
Article
Although gene therapy and DNA vaccination suggest promising new approaches to disease treatment and nonviral vectors (which are cheap and easy to manufacture) afford low immunogenicity, better safety profiles, and improved - commercial-scale purification of plasmid DNA remains difficult, particularly if bovine-derived ribonuclease A, is left out of the process. This article. series reviews the benefits and limitations of current plasmid DNA purification and suggests an RNase-free downstream process that is scalable, robust, and meets the requirements set by industry regulators.
Article
We describe and discuss a simple dry-compression technique for preparing a flat cuboid chromatography device containing a shallow packed-bed of crystalline hydroxyapatite nanoparticles. We then discuss the use of this device for fast protein separation in the bind-and-elute mode. Such separation could be carried out a quite low pressures, making it possible to use inexpensive low pressure chromatography systems. In the flow rate range examined in this study, the pressure-drop across the device increased linearly with flow rate, indicating negligible media compaction during use. Using this device, binary protein mixtures could be separated in about a minute. Contrary to that observed in most packed-bed chromatographic separations, the width of the flow through and eluted peaks decreased with increase in flow rate. Therefore, both productivity and purity could be simultaneously maximized. The suitability of using this device for preparative protein separations was demonstrated by carrying out purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens the possibility of developing dry-compression based flat cuboid packed-bed chromatography devices for fast preparative protein separation.
Chapter
A major consideration in creating and analysing hybrid plasmids is to adopt a strategy that will minimize the amount of work involved in achieving a particular goal. The plasmids can be naturally occurring or can be recombinants constructed in vitro from restriction endonuclease-cleaved fragments ligated into a suitable cloning vector. In the case of naturally occurring plasmids, the aim of the analysis may be to determine their size and restriction maps, to examine whether they carry particular sequences, and to localize on the plasmid genomes the regions of interest. This chapter describes the analysis of hybrid plasmids. The methods useful for the analysis of large and low copy number plasmids, which are more likely to occur among naturally occurring plasmids, are also discussed. The chapter outlines the various steps in clone analysis, reviewing recent developments and describing some of the standard methods that are in current use.
Article
This chapter discusses the preparation and screening of a complementary DNA (cDNA) clone bank. The DNA copy of an mRNA molecule is termed cDNA. The term cDNA clone is in general used to describe a bacterial cell transformed by a plasmid containing the DNA copy of an RNA molecule. The preparation of such a recombinant plasmid normally involves the synthesis from the RNA of a double-stranded DNA copy that is then integrated into a restriction enzyme cleavage site within the plasmid molecule. Most of the cDNA clones that have been isolated contain DNA copies of eukaryotic messenger RNA (mRNA) sequences. The typical eukaryotic cell contains many thousands of different mRNA sequences. A complete “cDNA clone bank” from such a cell is a population of bacterial transformants, each containing a plasmid with a single cDNA insert, and with a sufficiently large number of individual transformants such that every mRNA molecule is represented at least once in the bacterial population.
Article
The entire genome of human hepatitis B virus (HBV) occurring in Latvia was sequenced. This sequence, which is 3182 nucleotides long, was compared with the other previously published HBV genomes and was shown to share maximum homology with HBV subtype ayw DNA. The coordinates of 4 main open reading frames as well as hairpin structures are very well conserved in the two genomes. The distribution of nucleotide substitutions among different HBV genomes suggests that the open reading frames P and X can fulfil a coding function. On the basis of primary stucture comparison for hepadnaviral DNAs several evolutionary conclusions can be drawn.
Article
This study was carried out to investigate plasmid amplification and other differentiation by microwave irradiation (1 mV/cm2) and chloramphenicol treatment (170 μg/ml) in Bacillus subtilis. The effect of microwave on B.subtilis was observed and the amounts of DNA, RNA, protein and plasmid were determined during the experiments. The data obtained were evaluated statistically and the variability of the amounts of DNA, Protein, RNA, Growth and plasmid exposed to microwave were less than those treated with chloramphenicol and these amounts significantly differ between microwave and chloramphenicol. Microwave exposure induces any broken fragment and the amounts of plasmids increased.
Article
We examined the stability of plasmid RP4 and its expression of antibiotic-resistance genes in suspended and sorbed Pseudomonas putida in aquifer microcosms. Test tubes containing different proportions of sterilized aquifer soil and groundwater were inoculated with bacteria and incubated for up to 26 d. Serial dilutions were made to agar plates with or without antibiotics, to quantify the functional stability of the plasmid. The structural integrity of RP4 was examined by plasmid extraction, digestion with restriction enzymes, and agarose gel electrophoresis. The plasmid-borne resistance gene expression disappeared in 80–90% of the cells during day 1 of incubation in aquifer soil and then remained at that frequency throughout the experiment. The RP4 plasmid was present in cells without antibiotic-resistance gene expression, indicating that the observed loss of plasmid-encoded activity was most likely due to a reduction in expression of the resistance genes. The increased growth rate in groundwater amended with glucose and phosphate had no significant influence on plasmid loss or antibiotic-resistance expression, suggesting that plasmid loss and antibiotic-resistance expression were independent of the growth rate. Most of the reduction of resistance gene expression was associated with the presence of soil particles, and 70% of the resistance expression was retained in bacteria incubated for 1 d in groundwater alone. Bacteria sorbed to the soil particles had a lower frequency of expression of resistance genes than suspended bacteria, but the difference was not caused by sorbed inorganic or organic chemicals. Resistance gene expression was partly recovered in suspended bacteria after in vitro exposure to the antibiotics and after first isolating on agar without antibiotics and then replica plating to agar containing the antibiotics.
Article
Efficient loading on a chromatographic column is the dilemma facing engineers of the process development in plasmid DNA purification. In this research, novel arginine affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, supercoiled plasmid could successfully be purified from clarified lysate by two stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47mmol/L exhibited the highest recovery yield and a much higher productivity among arginine affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of supercoiled plasmid from clarified lysate.This article is protected by copyright. All rights reserved
Article
A common technique for small-scale isolation of genomic DNA is via adsorption of the DNA molecules onto a silica scaffold. In this work, the isolation capacities of calcium aluminate based glasses were compared against a commercially available silica scaffold. Silica scaffolds exhibit a negative surface at the physiological pH values used during DNA isolation (pH 5-9), while the calcium aluminate glass microspheres exhibit a positive surface charge. Isolation data demonstrates that the positively charged surface enhanced DNA adsorption over the negatively charged surface. DNA was eluted from the calcium aluminate surface by shifting the pH of the solution to above its IEP at pH 8. Iron additions to the calcium aluminate glass improved the chemical durability without compromising the surface charge. Morphology of the glass substrate was also found to affect DNA isolation; 43-106μm diameter soda lime silicate microspheres adsorbed a greater quantity of genomic DNA than silica fibers with an average diameter of ∼2μm.
Article
DNA plasmid-based gene expression systems are being widely investigated for the potential treatment of genetic and acquired disease and for DNA-based vaccination. A number of human clinical trials are in progress using plasmid-based drugs. The regulatory framework that has been applied to biologicals such as recombinant DNA-derived proteins has proven to be generally applicable for regulating plasmid-based drugs as well. This was recently emphasized by the inclusion of therapeutic DNA plasmid products in the U.S. Food and Drug Administration's list of well-characterized biotechnology products. Present techniques for manufacturing and characterizing plasmids have been adapted from large-scale protein purification and from traditional molecular biology. Production of multi-gram quantities of plasmid, at purities of 95% or more, is currently possible, but further development of both manufacturing and analytical techniques is required. This review describes the approaches and methods currently used to manufacture and characterize DNA plasmids for pharmaceutical use, as well as recent changes in the regulatory environment that will impact future development and marketing of plasmids as human drugs.
Article
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The structural gene for plasmid-mediated ampicillin resistance resides upon a 3.2 X 10(6) dalton transposable sequence (TnA) flanked by short inverted repeated sequences that accompany its insertion. TnA was transposed to pMB8, a 1.8 X 10(6) dalton derivative of the colicingenic plasmid ColE1. Random deletions were introduced in the resultant 5 X 10(6) dalton recombinant plasmid by a combination of nuclease treatments in vitro. From this set of deletions a subset was isolated that contained deletions affecting the transposition of TnA. The deletions were mapped by digestion with restriction nucleases and electron microscopic analysis of DNA hetero-duplexes and were found to include one of the inverted repeated sequences or lie in the central portion of TnA. Complementation experiments were attempted between these plasmids and another compatible plasmid carrying a deletion in TnA that abolished its ampicillin resistance. The results of the deletion data indicate that approximately 2 X 10(6) daltons of TnA is required for transposition; the complementation experiments suggest that the terminal inverted repetition and the central region of TnA play different essential roles in TnA transposition.
Article
Full-text available
The electrophoretic behavior of closed circular, nicked circular, and linear duplex forms of bacteriophages φx174 and PM2, plasmid ColEl, and rat mitochondrial DNAs has been analyzed by agarose gel electrophoresis as a function of gel concentration, electric-field strength, and ionic conditions. The logarithm of electrophoretic mobility (μ) is a linear function of gel concentration (T) at all agarose concentrations and field strengths tested. The retardation coefficient (dμ/dT = KR) is characteristic of DNA conformation under controlled conditions. Alterations in electric-field strength and ionic conditions lead to predictable changes in the relative migration rates of the various DNA forms, thus defining conditions under which their separation can be optimized. The alkaline titration of a mixture of duplex conformational isomers was carried out on gels at neutral pH. Under conditions where closed circular DNA is stable to denaturation, separation of the linear and circular complementary strands of φX174, Coll, and PM2 was observed. Electrophoresis of closed circular DNA in the presence of the intercalating dye ethidium bromide provided an estimate of superhelix density for PM2 DNA in good agreement with values determined by other methods. In addition, the use of ethidium bromide offers additional control over conditions for optimizing separations of DNA conformational forms. At ethidium bromide concentrations above that necessary to remove all negative superhelical turns, the relative change in the electrophoretic mobility with increasing dye concentration appears to be distinguishably different for the closed circular, nicked circular, and linear duplex forms of PM2 DNA. The combined results of this investigation define several experimental conditions and strategies for the application of agarose gel electrophoresis to studies of DNA structure and function.
Article
Full-text available
A method of isolating circular plasmid DNA from cleared lysates of E.coli is described. Purification is achieved by virtue of the rapid re-annealing kinetics of supercoiled DNA. After a brief denaturation step, double stranded plasmid DNA is separated from denatured chromosomal DNA and RNA in a two-phase partition system using dextran and polyethylene glycol. The method is much more rapid than the conventional dyecentrifugation technique and plasmid DNA of comparalle purity and yield is obtained.
Article
The synthesis of β-galactosidase has been studied in a mutant strain of Escherichia coli K12, which is induced by heat treatment in buffer to synthesize β-galactosidase upon subsequent growth at a low temperature. The re-establishment of repression after heat treatment was almost completely inhibited by chloramphenicol, puromycin or methionine starvation and to some extent by 5-methyltryptophan. These results lead us to the conclusion that protein synthesis is required for the establishment of repression. A simple explanation of these findings is that the repressor of β-galactosidase synthesis is a protein, or has a protein component. The re-establishment of repression proceeded in the absence of required uracil when RNA synthesis was reduced to 4%. This result suggests only a small amount of RNA synthesis is involved in repressor formation.
Article
DNA fragments obtained from EcoRI endonuclease digestion of bacteriophage ϕ80pt190 (trp+) and the plasmid ColE1 were covalently joined with polynucleotide ligase. Transformation of Escherichia coli trp- strains to tryptophan independence with the recombined DNA selected for reconstituted ColE1 plasmids containing the tryptophan operon and the ϕ80 immunity region. Similarly, an EcoRI endonuclease generated fragment of plasmid pSC105 DNA containing the genetic determinant of kanamycin resistance was inserted into the ColE1 plasmid and recovered in E. coli. The plasmids containing the trp operon (ColE1-trp) and the kanamycin resistance gene were maintained under logarithmic growth conditions at a level of 25-30 copies per cell and accumulate to the extent of several hundred copies per cell in the presence of chloramphenicol. Cells carrying the ColE1-trp plasmid determined the production of highly elevated levels of trp operon-specific mRNA and tryptophan biosynthetic enzymes.
Article
Examination of the properties of ColE1 derivatives containing either deletions or insertions of transposable genetic elements, has enabled a functional map of plasmid ColE1 to be constructed.
Article
Escherichia coli strain SPA O converts methionine to ethylene by an inducible enzyme system. L-Cysteine, L-homocysteine, methionine derivatives and the sulphur-containing analogues of L-methionine also act as precursors of ethylene. Ethylene is produced by cell suspensions only in the presence of air; cell-free preparations can produce ethylene aerobically and anaerobically, but the extent to which they do so depends on the mode of culture growth. Light stimulates ethylene production by cell suspensions and its presence is essential for production by cell-free preparations. The kinetics of ethylene biogenesis and its pH and temperature optima suggest that ethylene is a secondary metabolite.
Article
Sublethally irradiated mice primed with dinitrophenyl (Dnp)-keyhole limpet hemocyanin immediately after irradiation or 30 days later and subsequently boosted with a second injection of antigen displayed a secondary response to Dnp characterized by antibody affinity greater than that in unirradiated controls. Also, in radiation chimeras primed with Dnp-keyhole limpet hemocyanin 120 days after syngeneic or allogeneic bone marrow transplantation the antibodies against Dnp produced after boosting were of higher affinity than the antibodies raised in normal mice. These findings are tentatively attributed to lack of suppressor thymus-derived lymphocytes (T cells) in sublethally irradiated mice and bone marrow chimeras, in which the enhanced ability to produce antibodies of high affinity may compensate for quantitative defects of the immune system.
Article
Structures resembling nuclei may be released by gently lysing human or frog cells in solutions containing non-ionic detergents and 1.95 M NaCl. These structures, or nucleoids, sediment in sucrose gradients containing intercalating agents in the manner characteristic of DNA that is intact, supercoiled and circular. They are depleted of nuclear protein and contain no endogenous RNA polymerase activity. We describe conditions for the transcription in vitro of these nucleoids by the RNA polymerase of Escherichia coli. We compared the kinetics of RNA synthesis directed by nucleoids and by DNA prepared using conventional procedures: nucleoids direct RNA synthesis at three to four times the rate of an equivalent weight of pure DNA and under appropriate conditions the DNA of each nucleoid can direct the synthesis of twice its own weight of RNA. Most of the newly-synthesized RNA remains within the nucleoid. Experiments with the inhibitor, rifampicin, reveal that all RNA synthesis depends upon the initiation of new RNA chains and that nucleoids and pure DNA possess similar numbers of initiation sites. RNA synthesis directed by nucleoids has unusual kinetics: maximal rates are attained only after a lag of about ten minutes. Almost no lag is seen with pure DNA. The lag is due to the slow rate of formation of complexes of polymerase with nucleoid DNA that can initiate. Removal of supercoils by irradiation with γ-rays or by the addition of the unwinding ligand, ethidium bromide, decreases the numbers of polymerase molecules able to initiate synthesis rapidly and increases the lag before maximal synthetic rates are achieved. Loss of super-coiling does not alter the maximum synthetic rate. Supercoiling in eukaryotic DNA clearly influences the initiation of RNA synthesis. These results are discussed with reference to the effects of supercoiling on the transcription of prokaryotic templates.
Article
Evidence is presented that in Xenopus laevis the 18S rRNA sequence is proximal to the 5' end of the rRNA precursor molecule and the 28S secquence is proximal to the 3' end. This assignment was made by digesting amplified ribosomal gene transcription complexes with EcoRI restriction endonuclease and spreading the cleaved transcription complexes for electron microscopy. From the known location of the EcoRI cutting sites within the transcribed region and the length of nascent chains on the cleaved transcription complexes, it was possible to make an unambiguous assignment of polarity.
Article
The plasmid pML 21, which was found to contain approximately 49% of the Col E1 genome was used to determine the template origin of single-stranded deoxyribonucleic acid (DNA) fragments (4 to 32% of the Col E1 units length) associated with Col E1 dna replicative intermediates. The results of DNA hybridization competition experiments indicate that the single-stranded fragments derive from the full length of the Col E1 DNA template as expected for Okazaki fragments and the plasmid pML 21 contains the replication origin of Col E1 DNA.
Article
Molecular studies of R factors of six groups, F(II), I(1), I(2), N, B, and H, defined on the basis of compatibility, support the conclusions drawn from genetic studies. In general, R factors of a given compatibility group are similar in size. Deoxyribonucleic acid (DNA) reassociation occurs freely between members of the same group but is minimal between heterologous groups. An exception to this was found in group H, of which one factor showed minimal homology with the remaining plasmids of the group. A further exception was found with groups I(1) and B, which, although genetically distinct, show between 18 and 28% of DNA homology. Groups I(1) and I(2) are molecularly distinct, despite the fact that they both stimulate the synthesis of I-fimbriae.
Article
A rapid method for the isolation of DNA from mouse cells grown in tissue culture is described. Cells are lysed in 0.24 m sodium phosphate, pH 6.8 buffer, containing 1% sodium dodecyl sulfate, 8 m urea, and 10−3m ethylene-diaminetetraacetic acid, and the crude lysate applied to a column of hydroxyapatite. RNA and proteins are removed from the column with 0.24 m sodium phosphate buffer containing 8 m urea while DNA is selectively eluted with 0.48 m sodium phosphate buffer. There is almost total recovery of cellular DNA from the column and the DNA is virtually free from proteins and RNA. This extraction procedure indicates that a mouse cell contains about 8 × 10−6 μg of DNA.
Article
An electrophoretic method has been developed for the analysis of ribonucleic acids (RNAs) ranging in size from 104 to 108 daltons. The method depends on the use of acrylamide gels strengthened with agarose for analysis of the larger RNAs. The resolving power of the method permitted individual characterization of RNAs in mixtures containing multiple species of RNA, without prior purification of each species; RNA molecules which differed in molecular weight by only a few per cent could be clearly distinguished, and the molecular weight of each estimated. This unusual application of electrophoretic methods for the determination of molecular weight is based on the observation that, for RNAs, smaller molecules migrate more rapidly than larger ones. The mobility and the logarithm of the molecular weight are inversely related and this relationship is approximately linear. The molecular weights estimated by this technique, although numerically dependent on values assigned to known RNA standards, are highly reproducible in gels of various composition, and are at present the best means of identification of species resolved by gel electrophoresis. By this means, liver 18S RNA is identified as a doublet of RNAs of 0.66 and 0.62 × 106 daltons and the analogous 16 S of Escherichia coli as a doublet of 0.58 and 0.54 × 106 daltons, while liver 5S RNA has a molecular weight of 38,000 daltons.
Article
Using the stepwise elution technique, native doublestranded DNA is eluted by 0.20 M and 0.25 M potassium phosphate buffer, pH 6.8: occasionally minor fractions are eluted by 0.30 M and 0.50 M phosphate. Rechromatography experiments showed that the multipeak pattern obtained is an artifact. Indeed, when using the gradient elution technique all native DNA preparations investigated in the present work were eluted as single peaks.A different chromatographic behaviour was shown by singlestranded DNA from Φ X174 phage, yeast mitochondrial DNA, and glucosylated DNA.No significant changes in the physical, chemical or biological properties of native DNA are caused by the adsorption-elution process. The native DNA's studied here were not fractionated by hydroxyapatite columns as far as their base composition and biological properties are concerned. A very limited extent of fractionation on the basis of molecular weight was observed. It was shown that elution can be performed at constant ionic strength and in the presence of a variety of organic molecules.
Article
The 23S twisted circular form of ColE(1) DNA has been isolated from Escherichia coli as a tightly associated DNA-protein complex with a sedimentation coefficient of approximately 24S. Treatment of this complex with pronase, trypsin, sodium dodecyl sulfate, Sarkosyl, or heat results in a conversion to a slower sedimenting form of 17S or 18S, as determined by centrifugation in neutral sucrose gradients. These treatments do not alter the sedimentation properties of noncomplexes supercoiled ColE(1) DNA even in the presence of the ColE(1)-protein complex. Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE(1)-protein complex after treatment with these various agents is largely owing to an induced transition of ColE(1) DNA from the supercoiled to the open circular state.
Article
Covalently closed circular duplex DNA's are now known to be widespread among living organisms. This DNA structure, originally identified in polyoma viral DNA,(1,2) has been assigned to the mitochondrial DNA's in ox(3) and sheep heart,(4) in mouse and chicken liver,(3) and in unfertilized sea urchin egg.(5) The animal viral DNA's--polyoma, SV40,(6) rabbit(7) and human(8) papilloma--the intracellular forms of the bacterial viral DNA's φX174,(9,10) lambda,(11,12) M13,(13) and P22(14) -- and a bacterial plasmid DNA, the colicinogenic factor E2,(15) have all been shown to exist as closed circular duplexes. Other mitochondrial DNA's(16,17) and a portion of the DNA from boar sperm(18) have been reported to be circular, but as yet have not been shown to be covalently closed.
Article
Methods for preparation of stable columns of calcium phosphate (hydroxylapatite) suitable for protein chromatography and conditions for specific elution of proteins on such columns have been worked out. Elution is brought about by stepwise or continuous increase in the concentration of the eluant (phosphate buffer of constant pH), or by displacement. A number of proteins of molecular weights from about 10,000 to several millions have been chromatographed. In several cases multiple components were observed, indicating inhomogeneity. Some characteristic features of the procedure have been discussed, particularly the narrow elution ranges and the marked mutual displacement effects shown by many proteins. Low-molecular-weight material was found to show little or no adsorption on these columns. A description is given of photographic and television methods of observing directly colorless protein zones on the columns, making use of their light absorption in the short-wave ultraviolet region.
Article
The use of ultraviolet absorption measurements for the estimation of RNA by the Schmidt-Thannhauser procedure has been examined on samples of rat liver. Considerable amounts of protein are released into the RNA fraction during prolonged digestion in alkali, and the consequent errors in ultraviolet absorption cannot be adequately corrected by taking readings at two wavelengths and applying equations. On the other hand, a 1-h period of digestion in alkali at 37° releases negligible quantities of protein, provided that the concentration of alkali is low. Recovery of RNA is not impaired by this shortening of the period of digestion. Lipids need not be removed from the tissue.
Article
Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins (l), a number of modified analytical pro- cedures ut.ilizing this reagent have been reported for the determination of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and in insulin (10). Although the reagent would seem to be recommended by its great sen- sitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard t.o effects of variations in pH, time of reaction, and concentration of react- ants, permissible levels of reagents commonly used in handling proteins, and interfering subst.ances. Procedures are described for measuring pro- tein in solution or after precipitation wit,h acids or other agents, and for the determination of as little as 0.2 y of protein.
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Our technique has now been successfully used for the isolation of plasmids from cleared lysates of Bacillus subtilis ( S . D. Ehrlich, personal communication) and Streptococcus luctis
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Note Added in Proof. Our technique has now been successfully used for the isolation of plasmids from cleared lysates of Bacillus subtilis ( S. D. Ehrlich, personal communication) and Streptococcus luctis (F. L. Davies, personal communication). It has also proved possible to isolate the large plasmids RP4 and RP4: Mp by this method (S. B. Primrose, unpublished observations).