Benzpyrene groups bind preferentially to the DNA of active chromatin in human lung cells.

The cells of the bronchial epithelium of man are targets for benzo(a)pyrene carcinogenesis. When cultures of these cells, and of non-target fibroblasts, are exposed to [3H]-benzo(a)pyrene, we find that the epithelial cells metabolise and bind to DNA far greater amounts of benzpyrene than do fibroblasts. By analysis of nuclei of benzpyrene-treated cells for sensitivity to limited digestion with pancreatic DNase I, we have shown that benzpyrene groups bind initially to the DNA of expressed (DNase I sensitive) regions of chromatin in both cell types. Covalent binding of benzpyrene groups to non-expressed (DNase I resistant) regions follows rapidly in the target epithelial cells. These maintain high levels of carcinogen adducts in their DNA. In fibroblasts, benzpyrene group binding to non-expressed DNA occurs more slowly and active removal of adducts from the DNA is evident.     

Affinity chromatography of RecA protein and RecA nucleoprotein complexes on RecA protein-agarose columns

We have analyzed the nature of RecA protein-RecA protein interactions using an affinity column prepared by coupling RecA protein to an agarose support. When radiolabeled soluble proteins from Escherichia coli are applied to this column, only the labeled RecA protein from the extract was selectively retained and bound tightly to the affinity column. Efficient binding of purified 35S-labeled RecA protein required Mg2+, and high salt did not interfere with the binding of RecA protein to the column. Complete removal of the bound enzyme from the affinity column required treatment with guanidine HCl (5 M) or urea (8 M). These and other properties suggest that hydrophobic interactions contribute significantly to RecA protein subunit recognition in solution. Using a series of truncated RecA proteins synthesized in vitro, we have obtained evidence that at least some of the sequences involved in protein recognition are localized within the first 90 amino-terminal residues of the protein. Based on the observation that RecA proteins from three heterologous bacteria are specifically retained on the E. coli RecA affinity column, it is likely that this binding domain is highly conserved and is required for interaction and association of RecA protein monomers. Stable ternary complexes of RecA protein and single-stranded DNA were formed in the presence of the nonhydrolyzable ATP analog adenosine 5'-O-(thiotriphosphate) and applied to the affinity columns. Most of the complexes formed with M13 DNA could be eluted in high salt, whereas a substantial fraction of those formed with the oligonucleotide (dT)25-30 remained bound in high salt and were quantitatively eluted with guanidine HCl (5 M). The different binding properties of these RecA protein-DNA complexes likely reflect differences in the availability of a hydrophobic surface on RecA protein when it is bound to long polynucleotides compared to short oligonucleotides.     

Affinity of interactions between human glucocorticoid receptors and DNA: at physiologic ionic strength, stable binding occurs only with DNAs containing partially symmetric glucocorticoid response elements

Sucrose density gradient shift assays were adapted to permit determination of the affinity of interaction between human glucocorticoid receptors (GR) and DNA under conditions of DNA excess. Saturation analyses were performed to ascertain dissociation constants for the interaction of activated human GR with each of five DNA fragments. Centrifugation of GR-DNA complexes on sucrose gradients under nearly isotonic salt conditions revealed similar affinities with dissociation constants in the range of 2-16 nM for GR interaction with DNA fragments containing glucocorticoid response elements (GREs) exhibiting partial dyad symmetry. By contrast, GR exhibited virtually no affinity for non-GRE-containing DNA or for DNA containing only GRE half-sites. Additionally, GR showed evidence of multiple-site interaction with a DNA fragment containing two partially symmetric GREs, but interacted at only one site of an MMTV LTR DNA fragment containing a single partially symmetric GRE along with a cluster of three half-GREs. Together these data indicate that under physiologically relevant conditions, glucocorticoid receptors have high selectivity and affinity only for DNA containing specific partially symmetric GREs and further suggest that this high affinity for such DNA sites may be sufficient to account for the selective regulation of gene expression observed in glucocorticoid-responsive cells.     

A simple purification of calmodulin by reversed phase chromatography with hydrophobic polymer 3520

A new adsorption chromatography procedure for the purification of calmodulin from bovine brain was developed using polymeric adsorbent 3520. Calmodulin was first isolated by DEAE-Cellulose column chromatography and further purified to apparent homogeneity following elution with 50% ethanol from the adsorbent column. Polyacrylamide gel electrophoresis showed one band either in the presence of Ca2+ or EGTA. The polymeric adsorbent 3520 is a non-polar polymer lacking exchangeable groups. The selective adsorption of calmodulin is based on hydrophobic interaction within the matrix, and is Ca2+ independent. Neither high salt (0.5 M NaC1) nor EGTA (5 mM) was able to elute the CaM from the adsorption column whereas ethanol (50%) eluted it completely. This method is simple to use and it provides highly purified calmodulin with high yield.     

Isolation of DNA-protein complexes based on streptavidin and biotin interaction

We describe a method for the purification of proteins binding to specific DNA sites based on the strong interaction between streptavidin and biotin. We tested the efficiency of this method using the Escherichia coli lactose operon operator-repressor system. dUTP coupled to biotin is incorporated into a DNA fragment containing the lactose operator. A crude E. coli extract is first incubated with the biotinylated fragment and the reaction mixture is filtered on a streptavidin-agarose column. Proteins retained on the column are either eluted alone by high salt or isopropyl beta-D-thiogalactoside, or as a complex with the DNA site by enzymatic digestion of the DNA. We thus obtained a 3400-fold enrichment of the repressor complexed to the operator in one step. The method is simple and makes use of commercially available reagents. The large concentration of biotin-binding sites of the streptavidin-agarose matrix (0.1 mumol/ml packed gel) provides a very high capacity for the concentration and purification of large amounts of proteins. The advantage of this method for the detection and purification of other DNA-binding proteins is discussed.     

Negative chromatography: Progress,applications and future perspectives

In negative chromatography, the impurities bind on the adsorbent, and the product is allowed to flow through the chromatographic column. Negative chromatography is an alternative to positive chromatography under certain circumstances and has been used to purify various biomolecules. For this review, a detailed survey of the performance of reported studies on negative chromatography was conducted. The applications of negative chromatography in the capture and intermediate purification steps for biomolecules (e.g., plasmid DNA, antibodies, enzymes, hemoglobin, virus particles and cells) are reviewed. The negative chromatographic adsorbents adsorb the impurities through surface charge, hydrophobic interaction at specific sites on the surface, hydrophobic interaction, hydrogen bonding and functional groups. Examples of applications of negative chromatography according to the type of chromatography matrix used are summarized and discussed. In addition, the effects of operating conditions (initial protein concentration, buffer ions, pH and salt concentration) are discussed, and the criteria for choosing negative or positive chromatography are summarized. The literature survey showed that there will be future limitations and challenges ahead in implementation of negative chromatography. Possible solutions to the limitations and challenges of negative chromatography and future trends for developing negative chromatography are discussed.     

Characterization of recombinant human granulocyte-colony-stimulating factor produced in mouse cells.

Mouse C127I cells were transformed with a chimeric plasmid consisting of bovine papillomavirus DNA and human granulocyte-colony-stimulating factor (G-CSF) cDNA placed under the control of the SV40 early promoter. The transformed cells secreted constitutively a high level of human G-CSF, 10-20 micrograms/ml in a low-serum medium. The secreted G-CSF has been purified to homogeneity by a two-step procedure including gel filtration and hydrophobic column chromatography. The purified recombinant G-CSF runs as a single band with an apparent Mr of 19,000 on a polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This value corresponds to that of the native human G-CSF purified from the medium conditioned by human carcinoma CHU-2 cells. The recombinant human G-CSF was as active as native G-CSF in vitro in supporting proliferation of mouse NFS-60 cells and stimulating colony formation from human as well as mouse bone marrow cells. When the recombinant human G-CSF was subcutaneously administrated into mice, a remarkable stimulation of granulopoiesis and splenomegaly was observed.     

费氏中华根瘤菌与耐盐有关的DNA片段的亚克隆和测序

将费氏中华根瘤菌（Ｓｉｎｏｒｈｉｚｏｂｉｕｍ ｆｒｅｄｉｉ）ＫＴ１９与耐盐有关的２３ｋｂ ＤＮＡ片段用ＢａｍＨⅠ酶切成大小不同的长度,分别与质粒ｐＭＬ１２２连接,然后转化大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ）Ｓ１７－１,筛选出３个转化子。以这些转化子为供体,ＲＴ１９的盐敏感突变株ＲＣ３－３为受体,分别进行二亲本杂交,筛选到接合子ＢＲ２,得到４．４ｋｂ与耐盐有关的ＤＮＡ片段。根据其物理图谱,酶     

Hydrophobic properties of porcine fibronectin and its functional domains

The relations between surface hydrophobicities and binding properties of the functional domains of porcine plasma fibronectin were investigated. Porcine plasma fibronectin as well as human plasma fibronectin was adsorbed on a hydrophobic column with butyl or phenyl ligands in the presence of 0.5 M ammonium sulfate, and recovered in a single peak by decreasing the concentration of ammonium sulfate to 0 M, indicating that both fibronectins have very high surface hydrophobicities. On digestion with thermolysin, porcine plasma fibronectin yielded five fragments (140-150, 43, 25, 17, and 14 kDa) similar to those reported for human fibronectin, although porcine fibronectin was more resistant to the digestion than human fibronectin. The three heparin-binding fragments were found to have a wide range of surface hydrophobicities, the 140-150 kDa fragment having the lowest, the 25 kDa fragment a higher, and the 14 kDa fragment the highest among all the fragments. The 43 kDa collagen-binding and 17 kDa fragments had surface hydrophobicities as high as that of fibronectin. It is noteworthy that the 43 kDa collagen-binding fragment contributes to the high surface hydrophobicity of intact fibronectin in spite of the high content of carbohydrates.     

Intracellular apoptosis-inducing factor is induced by a vacuolar type H+-ATPase inhibitor in B lineage cells

We previously reported that concanamycin A, a specific inhibitor of vacuolar type H+-ATPases, induces DNA fragmentation in B cell hybridoma HS-72 cells. In the present study, we found that the cytosol from concanamycin A-treated HS-72 cells had a cytotoxic effect on intact cells in a cell viability assay. While activin A also induced apoptosis in HS-72 cells, the cytosol from activin A-treated HS-72 cells had no effect on cell viability. We purified the cytosol from concanamycin A-treated HS-72 cells by a four-step procedure: ultracentrifugation; HiTrap heparin column chromatography; HiTrap Q column chromatography; and reverse-phase high performance liquid chromatography on a C18 hydrophobic support. The biologically active fraction, which was used as partially purified cytosol, gave a specific band of protein with a molecular mass of 33 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mechanism of cell death was examined by observing changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of degraded chromosomal DNA, indicating the induction of apoptosis in cells cultured with the partially purified cytosol. The overexpression of human Bcl-2 suppressed apoptosis, indicating that the cytosol from concanamycin A-treated HS-72 cells induces apoptosis by a Bcl-2-inhibiting mechanism. These findings suggest that concanamycin A, a vacuolar type H+-ATPase inhibitor, produces intracellular apoptosis-inducing factor in B cell hybridoma.     

Mouse DNA methylase: methylation of native DNA.

An improved method of purification of DNA methylase from Krebs II ascites cells is reported. The enzyme sediments at 8.3 S on glycerol-gradients and a major band on SDS polyacrylamide gel electrophoresis has a molecular weight of 184 000. Aggregation occurs at low salt and this may interfere with enzymic activity. The preferred double stranded DNA substrate is that rendered partially unmethylated by an in vitro repair mechanism or by isolation from methionine starved cells. Methylation of native partially methylated DNA is favoured under conditions of low salt and high temperature; conditions which encourage 'breathing' of the DNA. Methylation of native, unmethylated DNA also involves breathing but results in formation of a salt resistant tight binding complex between the enzyme and the DNA.     

Integrated fragmentation of human IgG and purification of Fab using a reactant adsorptive membrane bioreactor separator system

This article discusses an integrated separation–reaction–separation scheme for producing Fab fragment directly from human immunoglobulin G (hIgG) present in serum feed. The novel reactant adsorptive membrane bioreactor separator (or RAMBS) system used in the current study consisted of a stack of microporous adsorptive membranes held within a temperature controlled module. The membrane stack, in the presence of salt, selectively and reversibly adsorbed hIgG by hydrophobic interaction while allowing most other serum proteins to flow through. The bound hIgG was then fragmented by pumping a solution of papain through the reactor at controlled temperature and flow rate. The salt concentration and pH for reaction and separation were systematically optimized using pure hIgG as reactant. The Fab fragment was separated from undigested hIgG and other byproducts such as Fc fragment based on their differences in hydrophobicity. Under optimal conditions, Fab was obtained in the reaction flow through while the other proteins remained bound to the membrane, these being subsequently eluted by lowering the salt concentration. The RAMBS system in addition to being convenient from process integration and intensification points of view also showed higher catalytic efficiency of papain in comparison to that in liquid phase reactions. Biotechnol. Bioeng. 2009; 104: 152–161 © 2009 Wiley Periodicals, Inc.     

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.     

Improved methods for separation of human cytokeratins.

The cytokeratins from human bladder and esophageal epithelia were separated using chromatographic techniques. The cytokeratins were first extracted from fresh autopsy tissue using high and low salt buffers. Urea, 8.0-9.5 M, was used to solubilize the resulting cytokeratin pellet. Imidazole was found to increase the solubility of the pellet but reducing agents such as 2-mercaptoethanol were not beneficial. DEAE ion exchange chromatography produced three fractions which were analyzed by using one and two-dimensional electrophoresis. The third fraction was shown to contain the acidic cytokeratins and was further fractionated on a moderately polar reverse phase HPLC column using an acetonitrile elution gradient. Tetramethylammonium tetrafluoroborate was added to the mobile phase to react with any unreacted silanol groups on the stationary phase, and trifluoroacetic acid was added to ion pair with the protein. HPLC fractions of the acidic proteins from human esophagus revealed seven reproducible peaks. All seven peaks were shown by Western blotting to contain an epitope found on cytokeratin 13. The results suggest that the isolation and separation procedures have produced a series of peptide products which all retain a similar epitope but which vary significantly in their hydrophobic characteristics.     

Properties of the telomeric DNA-binding protein from Oxytricha nova

Telomeres of Oxytricha macronuclear DNA exist as discrete DNA-protein complexes. Different regions of each complex display characteristic DNA-protein interactions. In the most terminal region, binding of a 43- and a 55-kDa protein to the telomeric DNA appears to account for all the DNA-protein interactions that can be detected by chemical and nuclease footprinting. We have used gradient sedimentation and protein-protein cross-linking to establish that the 43- and 55-kDa proteins are subunits of a heterodimer. Both subunits are very basic, which is unexpected considering the resistance of the DNA-protein interaction to high concentrations of salt. It is extremely difficult to dissociate the two subunits either from telomeric DNA or from each other. Even after extensive treatment of protein preparations with nuclease, a fragment of the 3' tail from macronuclear DNA remains bound to the protein. A wide range of conditions was screened for dissociation of the subunits from the DNA and/or from each other. Dissociation was only obtained by using conditions that caused some inactivation of the DNA-binding capacity of the protein. The use of reagents that covalently modify sulfydryl groups during the purification procedure facilitates preparation of telomere protein with full DNA-binding activity.     

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.     

Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins

Synapsin I is a major neuron-specific phosphoprotein that is specifically localized to the cytoplasmic surface of small synaptic vesicles. In the present study, the binding of synapsin I to small synaptic vesicles was characterized in detail. The binding of synapsin I was preserved when synaptic vesicles were solubilized and reconstituted in phosphatidylcholine. After separation of the protein and lipid components of synaptic vesicles under nondenaturing conditions, synapsin I bound to both components. The use of hydrophobic labeling procedures allowed the assessment of interactions between phospholipids and synapsin I in intact synaptic vesicles. Hydrophobic photolabeling followed by cysteine-specific cleavage of synapsin I demonstrated that the head domain of synapsin I penetrates into the hydrophobic core of the bilayer. The purified NH2-terminal fragment, derived from the head domain by cysteine-specific cleavage, bound to synaptic vesicles with high affinity confirming the results obtained from hydrophobic photolabeling. Synapsin I binding to synaptic vesicles could be inhibited by the entire molecule or by the combined presence of the NH2-terminal and tail fragments, but not by an excess of either NH2-terminal or tail fragment alone. The purified tail fragment bound with relatively high affinity to synaptic vesicles, though it did not significantly interact with phospholipids. Binding of the tail fragment was competed by holosynapsin I; was greatly decreased by phosphorylation; and was abolished by high ionic strength conditions or protease treatment of synaptic vesicles. The data suggest the existence of two sites of interaction between synapsin I and small synaptic vesicles: binding of the head domain to vesicle phospholipids and of the tail domain to a protein component of the vesicle membrane. The latter interaction is apparently responsible for the salt and phosphorylation dependency of synapsin I binding to small synaptic vesicles.     

Tight interaction between densely methylated DNA fragments and the methyl-CpG binding domain of the rat MeCP2 protein attached to a solid support.

In a previous report we have found that a number of short DNA fragments methylated at CpG sequences bound more tightly to a methyl-CpG binding column than DNA fragments having a larger number of methyl-CpG sequences. The column consists of a polypeptide comprising the DNA binding domain of the rat MeCP2 protein attached to a solid support. In the present study, we have investigated the features of short DNA fragments which bind tightly to a methyl-CpG binding column. Tight binding was observed when the DNA fragment had a high density of methyl-CpG sequences. Many of these fragments, derived from human genomic DNA, contained Alu repeated sequences supporting the previous observation that the highly-abundant Alu sequences are highly methylated. Our results suggest that methyl-CpG density is an important factor in the interaction between DNA fragments and the DNA binding domain of MeCP2 attached to a solid support.     

苜蓿中华根瘤菌与耐盐有关DNA片段的亚克隆和测序分析

将苜蓿中华根瘤菌(Sinorhizobium meliloti)042B与耐盐有关的4kb ClaⅠ DNA片段克隆在pML122上,用HindⅢ酶切下其2.4kb DNA片段,回收后与pBBR1\|MCS2连接,然后转化大肠杆菌(Escherichia coli)DH5α,筛选到转化子GS2〖DK〗。将残留在pML122上16kb ClaⅠ\|HindⅢ DNA片段连同质粒一起回收,让其自连,转化大肠杆菌S17-1,得到转化子GS0。以GS0为供体,042B的盐敏感突变株GZ17为受体,进行二亲本杂交,没有得到接合子。以GS2为供体,GZ17为受体,在辅助质粒pRK2013的协助下,进行三亲本杂交,筛选到接合子GG2,获得2.4kb HindⅢ与耐盐有关的DNA片段。将此片段连接到测序载体pGEM\|7Zf(+)上进行测序。测序结果表明,该24kb HindⅢ DNA片段含有3个开放阅读框(ORF)。在此基础上再一次亚克隆,获得19kb与耐盐有关的DNA片段。