Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction-modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.     

Modified DNA fragments activate NaeI cleavage of refractory DNA sites.

Endonuclease NaeI cleaves DNA using a two-site mechanism. The DNA-binding sites are nonidentical: they recognize different families of flanking sequences. A unique NaeI site that is resistant to cleavage resides in M13 double-stranded DNA. NaeI can be activated to cleave this site by small DNA fragments containing one or more NaeI sites. These activators are not practical for genetic engineering because unphosphorylated activators that are consumed during the cleavage of substrate give ends that may interfere with subsequent ligations. We show that a DNA fragment containing phosphorothioate linkages at the NaeI scissile bonds (S-activator) is not cleaved by NaeI, even though this S-activator binds to the substrate site. The S-activator activates NaeI to cleave M13 DNA under conditions that completely exhaust unsubstituted activator. These results demonstrate that activation is not coupled to cleavage of activator, that NaeI reverts to its inactive state soon after dissociation of the EA complex, and that S-activator makes for a nondepletable activator during prolonged incubations.     

Dinucleoside monophosphates. I. Optical properties and conformation in solution with one base charged.

A least squares analysis of the titration properties of several dinucleoside monophosphates enables calculation of the pK's for protonation. These pK's are used to resolve the spectral properties of dinucleoside monophosphates with one base charged from the apparent spectral properties of a dinucleoside monophosphate in aqueous solution. This method is applied to dinucleoside monophosphates containing adenosine and/or cytidine. Results of CD, nmr, and CD-temperature dependence measurements are presented. The results indicate that singly protonated dimers of these nucleosides stack as do their unprotonated analogs. It is suggested that this is true for all dimers with one base charged.     

The effect of cyclic anaerobic–aerobic conditions on biodegradation of azo dyes

The effect of cyclic anaerobic–aerobic conditions on the biodegradative capability of the mixed microbial culture for the azo dye Remazol Brilliant Violet 5R (RBV-5R) was investigated in the sequencing batch reactor (SBR) fed with a synthetic textile wastewater. The SBR had a 12-h cycle time with anaerobic–aerobic periods of 3/9, 6/6 and 9/3 h. General SBR performance was assessed by measurement of catabolic enzymes (catechol 2,3-dioxygenase, azo reductase), chemical oxygen demand (COD), color and amount of aromatic amines. In this study, under steady-state conditions, the anaerobic period of the cyclic SBR was found to allow the reductive decolorization of azo dye. Longer anaerobic periods resulted in higher color removal efficiencies, approximately 71% for the 3-h, 87% for 6-h and 92% for the 9-h duration. Total COD removal efficiencies were over 84% under each of the cyclic conditions and increased as the length of the anaerobic period was increased; however, the highest color removal rate was attained for the cycle with the shortest anaerobic period of 3 h. During the decolorization of RBV-5R, two sulfonated aromatic amines (benzene based and naphthalene based) were formed. Additionally, anaerobic azo reductase enzyme was found to be positively affected with the increasing duration of the anaerobic period; however; it was vice versa for the aerobic catechol 2,3-dioxygenase (C23DO) enzyme.     

Structure of NaeI-DNA complex reveals dual-mode DNA recognition and complete dimer rearrangement

NaeI, a novel DNA endonuclease, shows topoisomerase and recombinase activities when a Lys residue is substituted for Leu 43. The NaeI-DNA structure demonstrates that each of the two domains of NaeI recognizes one molecule of DNA duplex. DNA recognition induces dramatic rearrangements: narrowing the binding site of the Topo domain 16 A to grip DNA, widening that of the Endo domain 8 A to encircle and bend DNA 45 degrees for cleavage, and completely rebuilding the homodimer interface. The NaeI-DNA structure presents the first example of novel recognition of two copies of one DNA sequence by two different amino acid sequences and two different structural motifs in one polypeptide.     

Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.     

Resolution of (±)‐β‐methylphenylethylamine by a novel chiral stationary phase for Pirkle‐type column chromatography

In this study, a new Pirkle‐type chiral column stationary phase for resolution of β‐methylphenylethyl amine was described by using activated Sepharose 4B as a matrix, L ‐tyrosine as a spacer arm, and an aromatic amine derivative of L ‐glutamic acid as a ligand. The binding capacities of the stationary phase were determined at different pH values (pH = 6, 7, and 8) using buffer solutions as mobile phase, and enantiomeric excess (ee) was determined by HPLC equipped with chiral column. The ee was found to be 47%. Chirality, 2010. © 2009 Wiley‐Liss, Inc.     

Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation

Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, γ-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera^® (γ-Zein ER-accumulating domain) is the N-terminal proline-rich domain of γ-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera^® domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera^® domain. We demonstrated that Zera^®-F1-V protein accumulation was at least 3× higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera^® technology to induce protein body formation in non-seed tissues. Zera^® expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera^® technology to substantially increase the accumulation of value-added proteins in plants.     

Persistence of hyperbaric oxygen-induced oxidative effects after exposure in rat brain cortex tissue

Hyperbaric oxygen (HBO) causes oxidative stress in several organs and tissues. Due to its high rate of blood flow and oxygen consumption, the brain is one of the most sensitive organs to this effect. Many studies have reported oxidative effects of HBO, but there is no comprehensive data about how long this effect persists. The aim of this study was to elucidate the duration of HBO-induced oxidative/antioxidant action. Male Sprague-Dawley rats were divided into 5 groups. Except for the controls, the animals were subjected to 100% oxygen for 2 h at 3 atm and differed from each other by the time to dissection after exposure that began at 30, 60, 90, or 120 min. Thiobarbituric acid-reactive substances (TBARS), as well as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity was determined in brain cortex tissue. Additionally, nitrite-nitrate (NO(x)) concentrations were measured. All measured parameters were found to be significantly increased 30 min after exposure. SOD and GSH-Px levels persisted at significantly high levels for 60 min. In conclusion, the oxidative effect of HBO was shown to persist only for 1 h. Further studies should be performed to elucidate the possible molecular interactions during this period.