Novel biomineralization for hydrogels: electrophoresis approach accelerates hydroxyapatite formation in hydrogels

As a first step toward hydroxyapatite (HAp) formation in agarose hydrogels, we have tailored the internal chemistry using an electrophoresis approach. HAp was formed using aqueous solutions of calcium chloride and disodium hydrophosphate, which were set in a conventional agarose electrophoresis apparatus. Calcium and phosphate ions provided cations and anions, respectively, and were shown to migrate into the agarose hydrogel toward the corresponding electrode side. HAp was formed after colliding with each ion. The time needed to reach complete HAp formation was 30 min, and 130 ng of HAp was formed in 1 mg of agarose hydrogel when the equilibrium swelling state was reached. The electrophoresis approach accelerated the HAp formation, and the linear velocity of 1 mm/min was shown to be roughly 15 times larger than that of simple diffusion (0.06 mm/min).     

The isolation and characterization of differentially phosphorylated fractions of phosphofructokinase from rabbit skeletal muscle.

A preparation of phosphofructokinase from rabbit skeletal muscle is described which exploits the association-dissociation properties of the enzyme. Phosphofructokinase to prepared is partially phosphorylated and may be fractioned into three distinct species with sedimentation coefficients of 30 S, 18 S and 13 S by chromatography of agarose gels, hydroxyapatite or DEAE-cellulose. Measurements of alkali-labile phosphate content (phosphoserine and/or phosphothreonine) show that fractions consisting almost exclusively of 30-S species and fractions consisting predominantly of 18-S and 13-S species contain approximately 0.15 and 0.29 mol of phosphate per phosphofructokinase monomer (Mr = 80000) respectively. The results are interpreted in terms of at least two 13-S components which differ in their phosphate contents and also in their self-association properties. The possible significance of phosphorylation is discussed.     

Hydroxyapatite chromatography of short single-stranded DNA

Short single-stranded segments of calf thymus DNA were obtained by random cleavage with DNAase I. After treatment with various concentrations of DNAase I, fragment sizes were estimated using the ratio of total to terminal phosphorus. DNA populations ranging from 4--180 bases were obtained. Fragments with lengths up to 1140 were generated by shearing in a Virtis homogenizer. The hydroxyapatite elution profiles of sized populations were determined by elution with phosphate gradients. A curve relating elution molarities to single-strand chain length was 'biphasic', with the elution molarity being extremely sensitive to chain lengths below 50 nucleotides but much less sensitive to chain lengths above 100 nucleotides. These results show that single-stranded fragments below 50 nucleotides elute from hydroxyapatite appreciably before high molecular-weight denatured DNA using phosphate gradients. This is an important consideration when using hydroxyapatite to fractionate DNA populations which contain short single strands.     

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb¹. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits². During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel''s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight³. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along⁴. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation⁵; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments       

A rapid method for the purification of extrachromosomal DNA from eukaryotic cells.

A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA, is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.     

一株新的溶磷草生欧文氏菌的分离、鉴定及其溶磷效果的初步研究

从湖南、湖北、云南等地磷矿开采场的土壤样品中筛选到一株溶磷能力较强的菌株P21,结合生理生化指标和16S rDNA序列分析鉴定其属于草生欧文氏菌菠萝变种(Erwinia herbicola var.ananas).该菌能溶解磷酸三钙、羟基磷灰石、磷酸铁、磷酸锌,其中对磷酸三钙和羟基磷灰石的每升液体培养基溶磷量(P2O5)分别高达1206.20mg、529.67mg.溶磷菌草生欧文氏菌菠萝变种P21对产地不同的8种磷矿石溶解能力不同,对云南晋宁和昆阳、四川雅安、江苏锦屏等地磷矿石有较强的溶解能力,每升液体培养基溶磷量分别为96.64mg、78.46mg、67.07mg、65.24mg,对其它产地的磷矿石溶解能力较差.实验表明,培养液的pH下降与溶磷菌P21的溶磷量无直接关系.     

Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA

We have adapted a subtraction hybridization technique involving photoactivatable biotin, streptavidin binding, and organic extraction for solution hybridization analysis of mammalian genomic DNA. By combining maximal hybridization conditions of high salt, dextran sulfate, and formamide with successive hybridization steps and sequence enrichment by agarose gel electrophoresis, up to 97% of tracer DNA can be reproducibly driven to hybridize with photobiotinylated driver DNA. We demonstrate that the fractionation of hybridized from unhybridized sequences by this technique differs from hydroxyapatite chromatography with respect to the handling of nondenatured tracer, foldback sequences, and tracer-tracer hybrids. Strategies are presented to control the contribution of these species to the final subtracted product thereby making this technology a useful adjunct to solution hybridization approaches such as deletion cloning.     

Restriction fragment primed phi X174 single-stranded DNA as template for DNA polymerase alpha and beta. Detection and partial purification of a polymerase alpha stimulating factor

Template-primers constructed of phiX174 single-stranded viral DNA hybridized to a restriction fragment of phiX174 RF DNA can be used for extensive polymerization by DNA polymerase alpha. Polymerization is dependent upon a restriction fragment containing a 3'OH. The products of the reaction have been identified by agarose gel electrophoresis. Polymerization of 150--400 nucleotides can be obtained in 1h depending upon the restriction fragment used as primer. Synthesis may be limited by barriers in the primary or secondary structure of the template. A factor which stimulates the rate of alpha polymerase activity on these templates was partially purified. This factor does not stimulate alpha polymerase on activated DNA. The stimulating factor sediments at 5.5 S in glycerol gradients containing 0.4M potassium phosphate and has an apparent molecular weight of 70 000 on Sephadex G-100.     

Mechanism of action of nitroimidazole antimicrobial and antitumour radiosensitizing drugs. Effects of reduced misonidazole on DNA.

Electrolytic reduction of the hypoxic tumour cell radiosensitizing drug misonidazole was carried out at a controlled potential under anaerobic conditions in the presence of Escherichia coli DNA. During the reduction process the DNA was examined by viscometry, thermal hyperchromicity, melting and renaturation profiles, hydroxyapatite chromatography, agarose gel electrophoresis and alkaline sucrose density gradient centrifugation. The reduced drug decreases the viscosity, hyperchromicity and renaturation of DNA. These effects are consistent with strand breakage of the molecule which was corroborated by finding an increase in the single-strand content of DNA, increased migration and loss of fluorescence intensity on agarose gels and sedimentation to a less dense region in alkaline sucrose density gradients. The results are discussed in relation to postulated mechanisms of the selective toxicity of the drug towards anaerobes and cytotoxicity of electron affinic radiosensitizers of hypoxic tumour cells.     

Interaction of rat liver dexamethasone-receptor complexes with DNA]

Rat liver glucocorticoid-receptor complexes (GRC) acquire the ability to bind to DNA in a high affinity manner after activation by heating or precipitation with (NH)2SO4. DNA is practically non-saturable by GRC in low salt buffers as well as in 0.15 M NaCl-containing buffer, although in the latter case the binding decreases approximately 3--5 times. GRC bind to homo- and heterologous prokaryotic DNA in a similar way; in both cases an addition of KCl (up to 0.15 M) to the medium is followed by the same decrease of the binding. This data suggest that the association of GRC with DNA observed in vitro is not accompanied by "recognition" of any certain DNA site. Besides DNA, activated GRC can associate with other polymers, charged positively (DEAE-cellulose) or negatively (RNA, polyvinylsulfate). GRC interact very weakly with neutral compounds of the cellulose type but are strongly adsorbed on hydroxyapatite. Hence the activated GRC can be considered as an amphoteric protein. Salt solutions provoke dissociation of the GRC-DNA triple complexes: a complete dissociation is observed in the presence of 0,4 M NaCl or 0,4 M sodium phosphate buffer (pH 6,9). Sodium phosphate buffer also elutes GRC from other sorbents such as DEAE-cellulose or hydroxyapatite. No significant dissociation of the GRC-DNA complexes is observed at sucrose concentration up to 2 M. The data obtained are indicative of an essential role of electrostatic forces for the interaction of GRC with DNA. The non-ionic detergent Triton X-100 at a concentration as low as 0,05% completely destroys the GRC-DNA triple complexes. The models explicating the selectivity of the genome activation by GRC without their "recognition" of any specific DNA sequences are proposed.     

Separation of T-even Bacteriophage Deoxyribonucleic Acid from Host Deoxyribonucleic Acid by Hydroxyapatite Chromatography

A simple and rapid method is described for separation of T-even bacteriophage deoxyribonucleic acid (DNA) from host (Escherichia coli) DNA by hydroxyapatite column chromatography with a shallow gradient of phosphate buffer at neutral pH. By this method, bacteriophage T2, T4, and T6 DNA (but not T5, T7, or lambda DNA) could be separated from host E. coli DNA. It was found that glucosylation of the T-even phage DNA is an important factor in separation.     

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography

Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date.     

Hyperpolymer formation during renaturation of DNA from genomes with different sequence organisation.

Hyperpolymer formation during the renaturation of DNAs from wheat, calf and E. coli was studied using hydroxyapatite chromatography, electron microscopy and S1 nuclease. Large hyperpolymers could not be eluted from hydroxyapatite with 0.5 M phosphate buffer at 60 degrees C. Large proportions of wheat and E. coli DNAs were incorporated into hyperpolymers when fragments 650 nucleotides long were renatured. A much smaller proportion of calf DNA was incorporated under equivalent conditions. Greater proportions of calf DNA accumulated in hyperpolymers only when longer fragments were incubated. Electron microscopy indicated no obvious differences in the basic structures of hyperpolymers formed by the three DNAs and confirmed the quantitative differences in hyperpolymer formation found by hydroxyapatite chromatography. It is concluded that the proportions and arrangement of the repeated sequences in the chromosomes of higher organisms determine the extent of rapid hyperpolymer formation during DNA renaturation in vitro.     

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). 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.     

DNA staining in agarose gels with Zn²+-cyclen-pyrene

A pyrene-labeled Zn2+-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (～300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn2+-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.     

Recycling agarose

The cost of agarose for DNA gel electrophoresis can be significant in largescale projects. The International Potato Center (CIP) laboratory in Lima-Perú reuses agarose to save money. Due to limited financial resources for research, not uncommon in Latin American laboratories, this practice satisfies a need to control expenses. Recycled agarose can be used for RAPD analysis, or other applications in which no further manipulation of the DNA is necessary. Here we describe an improved CIP method where large amounts of agarose are ultimately equilibrated in water and dried to a powder similar to the original agarose.     

一种从琼脂糖凝胶中回收DNA片段的简单电洗脱装置

我们设计了一种简单电洗脱装置,从琼脂糖胶中回收ＤＮＡ。该装置由两个带旋盖的小管、两块透析膜和一个凝胶屏障组成。在电场作用下,ＤＮＡ从凝胶中迁移出来,通过凝胶屏障进入由凝胶屏障和透析膜组成的回收小仓。用微量吸样器收集ＤＮＡ,乙醇沉淀和清洗。该法ＤＮＡ的回收率约８５％;回收的ＤＮＡ可用于基因工程常规实验。     

DNA Staining in Agarose Gels with ZN2+-Cyclen-Pyrene

A pyrene-labeled Zn²⁺-cyclen complex for the staining of DNA in agarose gels is reported. The metal chelate coordinates reversibly to the DNA phosphate backbone, which induces the formation of pyrene excimers. The typical pyrene excimer emission is used for the detection of the DNA. Staining is limited to agarose gels and is less sensitive than ethidium bromide, but DNA amounts as low as 10 ng and short DNA strands (～300 b.p.) are detectable. Gel extraction as a standard technique in molecular biology was successfully performed after staining with Zn²⁺-cyclen-pyrene. Cytotoxicity tests on HeLa and V-79 cells reveal that the zinc-cyclen pyrene probe is significant less toxic compared to ethidium bromide.     

Studies on deoxyribonucleases from Haemophilus influenzae on DNA agarose affinity chromatography. Two-step purification of ATP-dependent deoxyribonuclease.

In a first part of this report, purification and characterization of several nucleased from lysates of Haemophilus influenzae are described. The enzymes bind to DNA with agarose columns and are removed by elution with phosphate buffer. Among the considered enzymes, the exonucleases 1 and 3, and endonuclease, a DNA polymerase and a restriction enzyme were recovered mixed by raising the phosphate concentration from 0.1 to 0.3 M, while the ATP-dependent DNAase recovered well purified, by raising the phosphate concentration to 0.45 M. After a rechromatography, on a second DNA with agarose column, of the peak of the ATP-dependent DNAase, the specific activity tested with 3H-labeled DNA was 125 units/mg of protein, representing a 300-fold purification of the original crude extract. In a second part, we have investigated the inactivation, at various pH, of transforming DNA of Haemophilus influenzae wild strain Rd with the different eluted fractions of the column, in order to determine the importance of contamination with other enzymatic activities, and also in order to confirm the nature of theisolated enzymes with a biological method. Finally, with enzymatic extracts of mutant strain Rd com minus 56, a strain which integrates shorter than normal pieces of DNA and which is suspected to possess and "activated specific endonuclease" able to recognize even small conformational modifications in paired structures, we tried to detect this activity on artificially constructed heteroduplex regions in DNA.     

SELECTIVE RETENTION AND FILTRATION OF BRAIN NUCLEIC ACIDS IN AGAROSE GELS

Abstract— Total nucleic acids of rat brain have been separated by agarose gel chromatography at 2 m -NaCl into DNA. transfer RNA plus low molecular weight RNA. and high molecular weight RNA fractions. The DNA fraction contained less than 1 per cent RNA by weight judged by either short-term or long-term labelling with ortho[³²P]phosphate. The high molecular weight RNA fraction contained 28 s and 18 s ribosomal RNAs and a heterogeneous population of 20-60 s RNAs, apparent after short-term labelling and characterized by a high content of nearest-neighbour-labelled uridylic acid. The rapidly sedimenting (>30 s ) portion of these RNAs could be largely separated from ribosomal RNAs by gel filtration using 4% agarose. The ribosomal RNAs could be fully resolved into 28 s and 18 s components by agarose gel chromatography at 0.5 m -0.6 m -NaCl, as shown by analysis of their sedimentation and nucleotide composition.