Studies on the selectivity of DNA precipitation by spermine.

We have examined the selectivity of the precipitation of DNA by spermine. We have found that the intra- and intermolecular condensation of DNA induced by spermine is highly selective even in the presence of added protein or triphosphates. We have also investigated the influence of buffer components on the threshold concentration of spermine required for DNA precipitation. Representative applications exploiting the selectivity of the precipitation reaction are also described.     

Selective regulation of S-adenosylmethionine decarboxylase activity by the spermine analogue 6-spermyne.

Polyamine-biosynthesis activity is known to be negatively regulated by intracellular polyamine pools. Accordingly, treatment of cultured L1210 cells with 10 microM-spermine rapidly and significantly lowered ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) activities in a sequential manner. By contrast, treatment for 48 h with 10 microM of the unsaturated spermine analogue 6-spermyne lowered AdoMetDC activity, but not ODC activity. An initial decrease in ODC activity at 2 h was attributed to a transient increase in free intracellular spermidine and spermine brought about through their displacement by the analogue. Thereafter, ODC activity recovered steadily to control values as 6-spermyne pools increased and spermidine and spermine pools decreased owing to analogue suppression of AdoMetDC activity. The apparent ability of 6-spermyne to regulate AdoMetDC, but not ODC, activity suggests an interesting structure-function correlation and demonstrates that the typical co-regulation of these enzyme activities can be dissociated. This, in turn, may reflect the existence of independent regulatory binding sites for the two enzymes.     

Selective chemical autoligation on a double-stranded DNA template.

We show that a double-stranded DNA segment serves as an effective template for spontaneously coupling short pyrimidine oligonucleotides containing terminal -P(O)(O-)S- and BrCH2C(O)NH- groups. The efficiency of this autoligation depends markedly on proper base-pairing between the probe oligomers and the double-stranded target. This chemistry should be useful in designing highly selective probes for double-stranded polynucleotide segments.     

Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high‐efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two‐stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion‐body stability is promoted by the addition of 15 mM 2‐hydroxyethyldisulfide (2‐HEDS), also known as oxidized β‐mercaptoethanol, to the permeabilization buffer (6 M urea + 3 mM ethylenediaminetetraacetate [EDTA]). 2‐HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion‐body fraction by centrifugation. In the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two‐stage process yielded an 81% (w/w) recovery of the recombinant protein Long‐R³‐IGF‐I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot‐scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 455–460, 1999.     

Identification of a protein cross-reacting with anti-phosphotyrosine antibodies in yeast insoluble cytoplasmic matrices

Immunoblot analysis with anti-phosphotyrosine antibodies of total extracts from exponentially growing yeast cells reveals a unique cross-reactive polypeptide of about 75 Kd (p75). The specificity of the immunodecorations has been checked by experiments of competition with phosphoaminoacids. A common behaviour has been observed for the 75 kd band and the 170 kd band corresponding to the platelet-derived growth factor receptor from Swiss 3T3 cells, which it has been known to be autophosphorylated on tyrosine upon ligand binding and used as a control throughout this work. We have found that p75 is associated to detergent insoluble cytoplasmic matrices. The stability of p75 detection by antibodies following treatments that specifically hydrolyze phosphohistidine and its susceptibility to potato acid phosphatase treatment provide further evidences that the epitope recognized by these antibodies in the yeast p75 polypeptide is indeed phosphotyrosine.     

DNA extraction by zinc.

A fast, very simple and efficient method of DNA extraction is described which takes advantage of DNA sedimentation induced by millimolar concentrations of ZnCl2. The zinc-induced sedimentation is furthermore strongly promoted by submillimolar phosphate anion concentrations. Within <30 min, the method recovers >90% of DNA irrespective of whether a plasmid DNA or short oligonucleotides are the extracted material. The method works with plasmid DNA and oligonucleotide concentrations as low as 100 ng/ml and 10 microg/ml, respectively, without using any expensive facilities or toxic chemicals.     

Selective colonization of insoluble substrates by human faecal bacteria

Insoluble plant polysaccharides and endogenous mucin are important energy sources for human colonic microorganisms. The object of this study was to determine whether or not specific communities colonize these substrates. Using faecal samples from four individuals as inocula for an anaerobic in vitro continuous flow system, the colonization of wheat bran, high amylose starch and porcine gastric mucin was examined. Recovered substrates were extensively washed and the remaining tightly attached bacterial communities were identified using polymerase chain reaction-amplified 16S rRNA gene sequences and fluorescent in situ hybridization. The substrate had a major influence on the species of attached bacteria detected. Sequences retrieved from bran were dominated by clostridial cluster XIVa bacteria, including uncultured relatives of Clostridium hathewayi, Eubacterium rectale and Roseburia species. Bacteroides species were also detected. The most abundant sequences recovered from starch were related to the cultured species Ruminococcus bromii, Bifidobacterium adolescentis, Bifidobacterium breve and E. rectale. The most commonly recovered sequences from mucin were from Bifidobacterium bifidum and uncultured bacteria related to Ruminococcus lactaris. This study suggests that a specific subset of bacteria is likely to be the primary colonizers of particular insoluble colonic substrates. For a given substrate, however, the primary colonizing species may vary between host individuals.     

Guanine modification during chemical DNA synthesis.

Base modification during solid-phase phosphoramidite synthesis of oligodeoxynucleotides has been investigated. We have discovered chemical modification that converts dG and dG-containing oligomers to a fluorescent form. This modification has been linked to N,N-dimethylaminopyridine (DMAP), an acylation catalyst, which can displace phosphate triester adducts at the 6-position of guanine. Further, we have found that this fluorescent intermediate can be converted in ammonium hydroxide solution to 2,6 diaminopurine deoxyribonucleoside (2,6 DAP), a potentially mutagenic nucleoside analog. We have shown that N-methylimidazole (NMI) in place of DMAP eliminates the fluorescent species and reduces 2,6 DAP contamination.     

Selective phosphorylation of human DNA methyltransferase by protein kinase C

Human DNA methyltransferase, the enzyme thought to be responsible for the somatic inheritance of patterns of DNA methylation, is an effective substrate for phosphorylation by protein kinase C. This provides a plausible mechanistic link between the action of tumor promoting phorbol esters, which stimulate protein kinase C, and abnormal patterns of DNA methylation often observed in transformed cells.     

Purification of recombinant protein A by aqueous two-phase extraction integrated with affinity precipitation

Aqueous two-phase extraction incorporated affinity precipitation was examined as a technique for protein purification. An enteric coating polymer, Eudragit S100, was employed as a ligand carrier. Eudragit was specifically partitioned to the top phase in the aqueous two-phase systems. For application of this method to purification of recombinant protein A using human IgG coupled to Eudragit in an aqueous two-phase system, 80% of protein A added was recovered with 81% purity. The purity was enhanced 26-fold by thid method. The IgG-Eudragit could be used repeatedly for the purification process. This seperation method should be applicable to industrial-scale purification as a new purification procedure combining the advantages and compensating for the disadvantages of the aqueous two-phase method and affinity precipitation method. (c) 1992 John Wiley & Sons, Inc.     

Selectivity of spermine homologs on triplex DNA stabilization.

We synthesized seven homologs of spermine (H2N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-9; n = 4 for spermine) and studied their effects on melting temperature (Tm), conformation, and precipitation of poly(dA).2poly(dT). The triplex DNA melting temperature, Tm1 was 34.4 degrees C in the presence of 150 mM KCl. Addition of spermine homologs increased Tm1 in a concentration-dependent and structure-dependent manner, with 3-6-3 (n = 6) exerting optimal stabilization. The dTm1/dlog[polyamine] values were 9-24 for these compounds. The duplex melting temperature, Tm2 was insensitive to homolog concentration and structure, suggesting their ability to stabilize triplex DNA without altering the stability of the underlying duplex. Circular dichroism spectral studies revealed psi-DNA formation in a concentration-dependent and structure-dependent manner. Phase diagrams were constructed showing the critical ionic/polyamine concentrations stabilizing different structures. These compounds also exerted structural specificity effects on precipitating triplex DNA. These data provide new insights into the ionic/structural determinants affecting triplex DNA stability and indicate that 3-6-3 is an excellent ligand to stabilize poly(dA).2poly(dT) triplex DNA under physiologic ionic conditions for antigene therapeutics.     

The interaction of spermine and pentamines with DNA.

We studied the effects of spermine, two naturally-occurring pentamines isolated from the thermophile Thermus thermophilus and one synthetic pentamine on the aggregation and 'melting' temperature of calf-thymus DNA and on the B-to-Z transition of poly(dG-me5dC). All pentamines caused aggregation of DNA at much lower concentrations than that of spermine. Concentrations that increased the melting temperature of DNA and induced the B-to-Z transition in poly(dG-me5dC) were different for each pentamine, but were comparable with the concentration of spermine needed to cause these effects. Our results suggest that both the total charge and the distance separating the charge, which is a function of the length of the carbon chains between amino groups, are important for the induction of conformational changes in DNA. The biological role of pentamines in T. thermophilus appears to be related to their ability to promote DNA condensation at high temperature.     

Selective export of HLA-F by its cytoplasmic tail

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.     

Polyethyleneimine-mediated chemical extraction of cytoplasmic His-tagged inclusion body proteins from Escherichia coli

The selectivity of polyethyleneimine (PEI) in DNA precipitation during chemical extraction was investigated. Chemical extraction was used to recover two His-tagged model proteins: gloshedobin, a thrombin-like enzyme from snake venom, and IbpA, a molecular chaperone, which were expressed mainly in the form of inclusion bodies. High DNA removal efficiency (more than 90%) was achieved at various cell densities (with OD600 ranging from 30 to 150) without affecting the solubility of host cell proteins. Compared to spermine-induced precipitation method reported elsewhere, PEI provided a higher DNA precipitation efficiency at a significantly lower cost. Moreover, PEI obviated the use of EDTA, which has been reported to be essential for the chemical extraction methods, hence exhibiting dual roles in replacing cost-prohibitive spermine and EDTA. The residual PEI in the post-extraction mixture was efficiently counteracted by addition of Mg2+, allowing the streamlined application of the extraction broth to immobilized metal affinity chromatography. Taken together, the PEI-mediated chemical extraction method provides a simpler and more economically viable processing route for the production of recombinant proteins whose expression is hampered by IB formation.     

Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.

Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents. Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli. We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks. dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide. In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles. In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps. The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.