Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I.

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.     

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris–HCl buffer [pH 8.0] containing 5 mM CaCl₂) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2 mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase.     

Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase

A procedure for the removal of RNA and RNA fragments from large quantities of pBR322 plasmid DNA without the use of RNase is described. Sephacryl S-300 is employed for the separation of low-molecular-weight RNA from plasmid DNA molecules on the basis of gel filtration. The technique thus circumvents many of the dangers associated with treating plasmid DNA preparations with RNase. The procedure should be generally applicable to the purification of virtually any type of plasmid DNA isolated from a bacterial host.     

Calcium is not required for immulectin-2 binding, but protects the protein from proteinase digestion

Mammalian C-type lectins are calcium-dependent carbohydrate-binding proteins. They serve as cell adhesion molecules in cell-cell interactions, or function as pattern-recognition receptors in innate immunity. Calcium is a direct ligand for carbohydrate binding in mammalian C-type lectins such as mannose-binding proteins and macrophage mannose receptor. In the tobacco hornworm Manduca sexta, a group of lectins named immulectins have been discovered. Each immulectin contains dual carbohydrate-recognition domains. Previously, we showed that immulectin-2 (IML-2) binds to a bacterial lipopolysaccharide, and agglutination of Escherichia coli cells by IML-2 is calcium dependent. In this study, we demonstrated that IML-2 bound to bacterial lipid A, smooth and rough mutants of lipopolysaccharide, lipoteichoic acid and peptidoglycan, as well as to fungal mannan and beta-1, 3-glucan (laminarin and curdlan). Binding of IML-2 to microbial components was calcium independent, and was increased by addition of spermine, a polyamine. In addition, plasma IML-2 bound to mannan-agarose independent of calcium. But trypsin digestion of IML-2 was inhibited in the presence of calcium. Our results suggest that calcium is not required for IML-2 binding but protects IML-2 from trypsin digestion.     

Properties of a subtilisin-like proteinase from a psychrotrophic Vibrio species comparison with proteinase K and aqualysin I.

An extracellular serine proteinase purified from cultures of a psychrotrophic Vibrio species (strain PA-44) belongs to the proteinase K family of the superfamily of subtilisin-like proteinases. The enzyme is secreted as a 47-kDa protein, but under mild heat treatment (30 min at 40 degrees C) undergoes autoproteolytic cleavage on the carboxyl-side of the molecule to give a proteinase with a molecular mass of about 36 kDa that apparently shares most of the enzymatic characteristics and the stability of the 47-kDa protein. In this study, selected enzymatic properties of the Vibrio proteinase were compared with those of the related proteinases, proteinase K and aqualysin I, as representative mesophilic and thermophilic enzymes, respectively. The catalytic efficiency (kcat/Km) for the amidase activity of the cold-adapted enzyme against succinyl-AAPF-p-nitroanilide was significantly higher than that of its mesophilic and thermophilic counterparts, especially when compared with aqualysin I. The stability of the Vibrio proteinase, both towards heat and denaturants, was found to be significantly lower than of either proteinase K or aqualysin I. One or more disulfide bonds in the psychrotrophic proteinase are important for the integrity of the active enzyme structure, as disulfide cleavage, either by reduction with dithiothreitol or by sulfitolysis, led to a loss in its activity. Under the same conditions, aqualysin I was also partially inactivated by dithiothreitol, but the activity of proteinase K was unaffected. The disulfides of either proteinase K or aqualysin I were not reactive towards sulfitolysis, except under denaturing conditions, while all disulfides of the Vibrio proteinase reacted in absence of a denaturant. The reactivity of the disulfides of the proteins as a function of denaturant concentration followed the order: Vibrio proteinase > proteinase K > aqualysin I. The same order of reactivity was also observed for the inactivation of the enzymes by H2O2-oxidation, as a function of temperature. The order of reactivity observed in these reactions most likely reflects the accessibility of the reactive cystine or methionine side chains present in the three related proteinases, and hence a difference in the compactness of their protein structures.     

Human RNase 7: a new cationic ribonuclease of the RNase A superfamily

Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence. The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine–lysine– histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3’s cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.     

Magnetic proteinase K reactor as a new tool for reproducible limited protein digestion

As an aid to differentiating between the prion proteins Prp(c) and PrP(Sc), the preparation and use of immobilized Proteinase K (PK) is described. An accumulation of PrP(Sc) in the central nervous system is the one of the causes of neurodegenerative disease. Current routine diagnosis is based on the postmortem detection of the distinct neuropathological lesion profiles of CNS and by the presence of the PK-resistant core of the prion protein isolated from brain lysates. An assay with PK immobilized to magnetic -COOH micro- and nanoparticles can offer a convenient as well as economic method. The individual immobilization steps were verified by measuring the zeta potential of the particles. The stability of the newly developed PK magnetic reactor, observed during kinetics measurements, was highly satisfactory. The calculated values of the apparent Michaelis constant (4.25 mM for native enzyme and 1.28 mM for immobilized enzyme) were determined from Lineweaver-Burk plots. Human growth hormone was digested using the newly prepared magnetic PK reactor and MALDI-TOF-MS analysis of the digests showed satisfactory efficiency. Controlled digestion of PrP(c) from the Mov mouse cell line was demonstrated with Western blot detection.     

DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays

Mapping DNase I hypersensitive sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers and locus control regions. Although Southern blots are the traditional method of identifying DNase I hypersensitive sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNase I hypersensitive sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We used DNase-chip to identify DNase I hypersensitive sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We found that although most DNase I hypersensitive sites were present in both cell types studied, some of them were cell-type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.     

Identification of amino acid residues in the catalytic domain of RNase E essential for survival of Escherichia coli: functional analysis of DNase I subdomain

RNase E is an essential Escherichia coli endoribonuclease that plays a major role in the decay and processing of a large fraction of RNAs in the cell. To better understand the molecular mechanisms of RNase E action, we performed a genetic screen for amino acid substitutions in the catalytic domain of the protein (N-Rne) that knock down the ability of RNase E to support survival of E. coli. Comparative phylogenetic analysis of RNase E homologs shows that wild-type residues at these mutated positions are nearly invariably conserved. Cells conditionally expressing these N-Rne mutants in the absence of wild-type RNase E show a decrease in copy number of plasmids regulated by the RNase E substrate RNA I, and accumulation of 5S ribosomal RNA, M1 RNA, and tRNA(Asn) precursors, as has been found in Rne-depleted cells, suggesting that the inability of these mutants to support cellular growth results from loss of ribonucleolytic activity. Purified mutant proteins containing an amino acid substitution in the DNase I subdomain, which is spatially distant from the catalytic site posited from crystallographic studies, showed defective binding to an RNase E substrate, p23 RNA, but still retained RNA cleavage activity-implicating a previously unidentified structural motif in the DNase I subdomain in the binding of RNase E to targeted RNA molecules, demonstrating the role of the DNase I domain in RNase E activity.     

DNase I hypersensitivity in the gamma globin gene locus of K562 cells.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.     

Preparation and properties of a new DNase from Aspergillus oryzae.

A DNase present in commercial preparations of Aspergillus oryzae alpha-amylase was purified 1550-fold in 25% yield by acetone precipitation and by chromatography on diethylaminoethyl- and carboxymethylcellulose. The enzyme was isolated free of contaminating RNases and DNases. The molecular weight of the enzyme determined by gel filtration on Sephadex G-100 was 48 000, while a molecular weight of 58 000 was determined for the single band observed upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The isoelectric point of the DNase is 9.2. The enzyme hydrolyzed only DNA with a pH optimum of 8.2 and was activated by Co2+, and to a lesser extent by Mg2+ and Mn2+. Native DNA was a better substrate than heat-denatured DNA. Enzymatic digests of calf thymus and E. coli DNA yielded oligomers of chain lengths ranging from 10 to 200, with mono- and small oligonucleotides (chain length less than 5) detected only when large (100 mg) amounts of DNA were fractionated by column chromatography on diethylaminoethyl-Sephadex A-25 in 7 M urea. The digestion products contained 5'-terminal phosphate groups and mostly adenosine at the 3' and guanosine and adenosine at the 5' ends.     

Calcium dependent cysteine proteinase is a precursor of a chemotactic factor for neutrophils

A new chemotactic factor for neutrophils is generated from calcium dependent cysteine proteinase (calpain) I by autodigestion. An active peptide was isolated from the autodigest and its structure was determined to be an acetylated nonapeptide with the sequence: N-acetyl Ser-Glu-Glu-Ile-Ile-Thr-Pro-Val-Tyr. Compared with the entire sequence of human calpain I, the peptide was identical with the N-terminal amino acid sequence of the large subunit deduced from the cDNA sequence, except that the peptide was devoid of a methionine residue and acetylated at the N-terminus. The acetyl nonapeptide was synthesized and its chemotactic activity was reconfirmed. The biological significance and possible role of this calpain derived chemotactic factor in inflammation are discussed.     

Purification and properties of a new DNase activity from KB cells.

A deoxyribonuclease activity with specificity towards single-stranded DNA has been purified approximately four hundred-fold from KB cells, by chromatography on DEAE-cellulose, phosphocellulose and hydroxylapatite. The last step of the purification results in separation of the enzyme from a DNase activity which has been described previously (Wang, E.C., Furth, J.J. and Rose, J.A., (1978) Biochemistry 17: 544-549). The properties of the new DNase activity are significantly different from those of the enzymes which have previously been identified in these cells. The activity sediments at approximately 7.5S in a glycerol gradient. The DNase activity is optimal at pHs between 6.0 and 6.5. It cleaves DNA endonucleolytically and hydrolyzes single-stranded DNA at about 11 times the rate of double-stranded DNA and at twice the rate of Poly (dA). The activity is moderately sensitive to inhibition by N-ethylmaleimide and is inhibited 80% by 50 mM NaCl. It is stimulated twenty-fold by Mn++ at an optimal concentration of approximately 0.7 mM. It is stimulated by a lesser extent by Mg++, but not by Ca++.     

Sugaring out: A new method for removal of acetonitrile from preparative RP-HPLC eluent for protein purification

Reverse phase-high pressure liquid chromatography (RP-HPLC) with an acetonitrile–water mixture as the eluent is widely used for purification of proteins. The separation of acetonitrile (ACN) in RP-HPLC eluent is important for protein recovery. Cooling below subzero temperature and salting out have been used to remove ACN, each with its limitations. In this work we have explored the use of sugaring-out, a new phase separation method developed at University of Illinois for the separation of ACN from a simulated preparative RP-HPLC effluent. The effect of glucose concentration, temperature, and initial amount of ACN in the effluent on phase separation was investigated. Results showed that a good phase separation can be achieved at near room temperature (18 °C). With the optimized conditions, we found that more than 60% (w/w) of ACN was removed and more than 95% (w/w) of water-soluble proteins (bovine serum albumin, trypsin, and pepsin) were recovered.